J. Phy8iol. (1976), 257, pp. 449-470 With 5 text-figure8 Printed in Great Britain

449

AUGMENTATION: A PROCESS THAT ACTS TO INCREASE TRANSMITTER RELEASE AT THE FROG NEUROMUSCULAR JUNCTION

BY K. L. MAGLEBY AND JANET E. ZENGEL From the Department of Phy8iology and Biophy8ic8, Univer8ity of Miami School of Medicine, Miami, Florida 33152, U.S.A.

(Received 14 October 1975) SUMMARY

1. End-plate potentials (e.p.p.s) were recorded from frog neuromuscular junctions bathed in Ringer solution containing increased Mg and decreased Ca to reduce transmitter release. Conditioning and testing stimulation was applied to the nerve to study a previously uncharacterized process which acts to increase e.p.p. amplitudes. We will refer to this process as augmentation. 2. Following repetitive stimulation augmentation decayed approximately exponentially over most of its time course with a mean time constant of about 7 see (range 4-10 see) which is intermediate in duration between the time constants for 'the decay of facilitation and potentiation. 3. The magnitude of augmentation increased with the duration of the conditioning stimulation. Assuming a multiplicative relationship between augmentation and potentiation, values of the magnitude of augmentation ranged from 0 3 to 0-6 following 50 impulses at 20/sec to 0-5-7-8 following 600 impulses at 20/sec. (An augmentation of 0 3 and 7-8 would increase e.p.p. amplitudes 1-3 and 8-8 times, respectively.) 4. The time constant characterizing the decay of augmentation remained relatively constant as the duration of the conditioning stimulation was increased. 5. Augmentation as well as facilitation and potentiation resulted from an increase in the number of quanta of transmitter released from the nerve terminal. 6. Augmentation decayed faster at higher temperatures with a mean temperature coefficient, Q10, of about 3-8. The corresponding Q10 for the decay of potentiation was found to be about 2'4. 7. It is concluded that augmentation can be a significant factor in increasing transmitter release and will therefore have to be accounted for when studying the effects of repetitive stimulation on the function of the nerve terminal or when formulating models of transmitter release. 17-2

450

K. L. MAGLEBY AND JANET E. ZENGEL INTRODUCTION

The end-plate potential (e.p.p.) produced by a testing nerve impulse may be increased in amplitude for seconds to minutes following repetitive stimulation (Feng, 1941; Liley, 1956; Hubbard, 1963; Braun, Schmidt & Zimmermann, 1966; Mallart & Martin, 1967; Rosenthal, 1969; Hubbard, Jones & Landau, 1971). At least two processes are involved in this increase: facilitation which has a time course of less than a second and potentiation which has a time course of tens of seconds to minutes (Landau, Smolinsky & Lass, 1973; Magleby, 1973b). Recently we presented evidence for a previously uncharacterized process which increased e.p.p. amplitudes following repetitive stimulation and decayed with a time course intermediate in duration between the time courses of facilitation and potentiation (Magleby & Zengel, 1975a). The purpose of this paper is to characterize this process further. The name augmentation seems appropriate for this process since it is found that it augments transmitter release and since it is not yet known whether its mechanism is similar to the mechanism of facilitation or potentiation, or whether it is a different process. Whatever the case, this paper shows that augmentation can be a significant factor in increasing transmitter release. Therefore studies on the effects of repetitive stimulation on the function of the nerve terminal and models for transmitter release will have to account for augmentation. In a preliminary report of some of these results augmentation was referred to as the intermediate process (Zengel & Magleby, 1975). METHODS

The results reported here and in the following paper (Magleby & Zengel, 1976) are based upon observations made in more than 100 sartorius nerve-muscle preparations from northern and southern varieties of the frog Rana pipiens and from five sartorius nerve-muscle preparations from the local toad Bufo marinu8. The results from these three types of preparations appeared qualitatively similar and have been combined in these papers. For most experiments extracellular recordings of end-plate potentials (e.p.p.s) were obtained with a surface electrode from end-plate regions. In a few experiments e.p.p.s were recorded with intracellular micro-electrodes using standard techniques. The experimental preparation and extracellular and intracellular recording techniques have been described in detail previously, where it was shown that surface recorded e.p.p. amplitudes give a good measure of the average intracellular activity (Magleby, 1973 a). The standard bathing solution used in these experiments had the composition (mm): NaCl 115, KC1 2, CaCl2 1-8, Na2HPO4 2-16, NaH2PO4 0-85, glucose 5, choline 0-03. This solution was modified by reducing Ca to 0-5-0-7 mm and adding 5 mm-Mg to greatly decrease transmitter release. The osmolarity of the solution was maintained by reducing NaCl. The solutions were adjusted to pH 7-27-4 before ilse. All experiments were done at 200 C unless otherwise indicated.

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In order to reduce depression due to possible depletion of transmitter (Thies, 1965), the data presented in this paper were collected under conditions of very low quantal content, and consequently, fluctuating transmitter release (del Castillo & Katz, 1954a). The surface electrode sums responses from many end-plates at once, but this averaging was not usually adequate to smooth the fluctuation in the response sufficiently to make accurate determinations of the magnitude and time courses of the processes under study from a single conditioning-testing trial. To overcome this problem, a variable number of trials (two to fifty) were averaged for each set of experimental conditions to obtain a better estimate of the mean response. Data were averaged only within a single neuromuscular preparation. The experiment was then repeated a number of times using a new neuromuscular preparation each time to obtain an estimate of the repeatability of the observations. Consequently, mean values and number of observations reported in this paper refer to the number of preparations in which the experiment was carried out. All experiments were done using a PDP-1I1 computer to generate the stimulation patterns, measure and store the e.p.p. amplitudes, and analyse the data during and following the experiments. Data were collected, stored, and analysed in terms of trials, each trial consisting of a control, conditioning, and testing sequence. In a typical trial the nerve was first stimulated 10 times once every 5 or 10 see to establish a control level of response. The nerve was then stimulated at a higher frequency (for example 200 impulses at 20/sec) to condition the nerve terminal. The effect of this conditioning stimulation was then determined by applying single testing impulses to the nerve. Usually three to six impulses were applied at 1-5-2 sec intervals immediately after the train to test for the fast decaying augmentation and then 50-100 impulses were applied at 5 or 10 sec intervals to test for the slower decaying potentiation. All e.p.p. amplitudes during the control, conditioning, and testing periods of the trial were measured and saved. Following each trial, the average of the first 10 and last 10 e.p.p. amplitudes of the trial, which gives an indication of any drift occurring in the preparation during the trial, were printed. All the e.p.p. amplitudes during the trial were then plotted against time on a storage oscilloscope to show the total response during the trial. The analysis and display of data after each trial took about 10 see, after which a new trial was started. A typical preparation could be run as long as 6-16 hr enabling the collection of over 50-100 trials. Signals from the recording electrodes were amplified and sent to an oscilloscope for monitoring and to the analogue-to-digital input ofthe computer. The analogue-todigital converter samples two channels simultaneously, one at normal gain and one at 1/8 the gain. The sample obtained from the 1/8 gain input was automatically used when the range of the normal input was exceeded. This arrangement made it possible using only a 12 bit A-D converter to sample e.p.p. amplitudes over a greater than 160 times dynamic range (200-32,760 bits) with less than 0-5 % error (excluding the error introduced by noise from the first high gain amplifier) in the estimation of the amplitudes. E.p.p.s were generated and measured as follows: when the programmed crystal clock in the computer indicated it was time to stimulate the nerve, three samples at the rate of 5/msec were taken to determine the base line voltage. The computer then generated a signal which was used to stimulate the nerve through a pulse generator and photo-isolation unit in the conventional manner. Following the shock artifact, twenty to sixty samples (depending on the temperature) were taken at a rate of 5/msec to record the e.p.p. The difference between the highest base line point and the highest peak point was then taken as the measure of the e.p.p. amplitude. The base line and e.p.p. sampling points and two horizontal lines representing the

452

K. L. MAGLEBY AND JANET E. ZENGEL

base line and the peak of the e.p.p. were displayed for every e.p.p. An example of such a display is shown in Fig. 1 B. The interval between e.p.p.s was always sufficient that it was not necessary to correct for one e.p.p. falling on the tail of a previous one. Thus, there was never any doubt of exactly what the computer was sampling and how the e.p.p. amplitudes were measured, for every e.p.p. was displayed and could be compared to the same data recorded and displayed on an oscilloscope in a conventional manner. After an experiment, each individual trial was usually displayed to check for consistency in the general form of the experimental data from trial to trial. In a few instances large unpredictable changes in e.p.p. amplitudes would occur within trials. This was assumed to result from nerve failure and these trials and those following were discarded. Following the checking of the individual trials, trials collected under the same experimental conditions and same general period of time were averaged with the computer and then displayed for comparison with data collected under different experimental conditions. Most of the Figures presented in this paper were made from data averaged in this manner. The decays of the e.p.p. amplitudes following the conditioning trains of the averaged data or of the individual trials, depending on the experiments, were then plotted semilogarithmically against time on the display monitor. Least squares fits to the experimental points within specified time ranges were then calculated and plotted. Estimates of potentiation were usually obtained by fitting a line to decay points between about 30 and 100 sec. Estimates of the magnitude and time course of augmentation were obtained by subtracting or dividing off the influence of potentiation asumn eter an additive or multiplicative relationship between augmentation and potentiation (Fig. 1). The derived experimental points for augmentation were then plotted and least-squares fits were made to both the additive and the multiplicative set of points falling be. tween about 1-5 and 20 sec. A complete analysis of the data, including inspection, averaging, and generation of five to twenty semilogarithmic plots with least-squares fits, could be accomplished in several hours. More than 2 x 106 e.p.p. amplitudes have been analysed in this manner. Intracellularly recorded e.p.p.s have been corrected for non-linear summation of unit potentials (Martin, 1955). No attempt has been made to correct extracellularly recorded data for non-linear summation of unit potentials. Consequently, e.p.p. amplitudes at the peak of the conditioning trains could be under-estimated by as much as 20-40 % in those experiments with high quantal. content, although intracellular studies tend to indicate that in most experiments the error would be less than 5-20 %. The error introduced in estimating augmentation and potentiation would be much less since the e.p.p. amplitudes used to estimate these processes are considerably smaller than those during the conditioning train (see Fig. 1 A). In those experiments where quantal contents were examined, the computer stimulated and sampled intracellularly recorded e.p.p.s as they were simultaneously recorded by an FM tape recorder. Individual trials were then played back from tape into a pen recorder at 1/10 of the recorded speed. Miniature end-plate potentials (m.e.p.p.s) were measured from the pen recorder record before, during, and following conditioning stimulation to look for possible changes in amplitude. The pen recordings were also examined to determine above what amplitude a response would be considered a success. When individual trials of intracellularly recorded e.p.p.s were displayed as e.p.p. amplitude against time, the amplitudes typically fell into quantal groups with an obvious separation between failures and successes. The size above which a response was considered a success determined in this manner agreed with that determined from the pen recording data of the trial. Quantal contents were then calculated by both the methods of failures and by dividing mean e.p.p. amplitude

AUGMENTATION OF TRANSMITTER RELEASE

453

by the mean m.e.p.p. amplitude (del Castillo & Katz, 1954a) for data collected before, during, and following repetitive stimulation. This was done by dividing each trial into segments of 5-100 successive nerve stimulations and then combining the same segments of data from each of 10-15 successive trials to obtain from 50 to 1500 responses to nerve stimulation for each determination of quantal content. Resting potentials were -85 to -95 mV on insertion of the micro-electrode and typically remained more negative than -80 mV during the several hours required to collect sufficient intracellular data from a single cell for the quantal content studies. For the experiments in which Q10 was estimated, five to ten trials were collected at one temperature; the temperature was then changed by heating or cooling the bottom of the preparation chamber with a Peltier device (the temperature change usually took about 5-10 min including a few minutes for the preparation to equilibrate at the new temperature.) Five to ten trials were then collected at this new temperature, after which the temperature was changed to the original temperature and 5-10 additional trials were collected. This pattern was repeated through two to ten temperature changes. The data were usually analysed in two ways: (1) by comparing the mean response of all the data obtained at one temperature to the mean response of all the data obtained at the other temperature, or (2) by comparing the mean response of the data collected in one group of consecutive trials collected at one temperature to the mean of the immediately preceding and following blocks of data collected at the other temperature. Q1.,s obtained in these two ways were similar, suggesting that the method of analysis or possible drift in the preparation had little effect on the results. The values of Q10 presented in this paper represent those obtained when the data were analysed by the method of comparing the mean responses of all the data collected at the two temperatures.

Terminology Facilitation, augmentation and potentiation are all defined in a similar manner; each one is given by the fractional increase of a test e.p.p. amplitude over a control in the absence of the other two processes, such that:

F(t)

v(t) ,AI(t) = lP(t)

A (t)

v(t) A

= 0 P(t) = 0

IF(t)

O

P(t)

0

=10

VO

I v(t) jOA(e) F(t)=O = 0 ,()

(1) 2 (2)

(3)

where F(t) is facilitation, A(t) is augmentation, P(t) is potentiation, v(t) is the e.p.p. amplitude at time t, and vo is a control e.p.p. amplitude when F(t), A(t) and P(t) all equal 0. Experimcntally, however, it is not usually possible to measure one process in the absence of the other two. Consequently the magnitude and time course of the individual processes are derived mathematically from the fractional change in e.p.p. amplitude which is given by V(t) = v(t) (4) V0

where V(t) is the fractional change in a testing e.p.p. amplitude at time t compared to a control e.p.p. amplitude. The experimentally determined estimation of the contribution that facilitation, augmentation, and potentiation make to increasing transmitter release during

K. L. MAGLEBY AND JANET E. ZENGEL

454

repetitive stimulation relies on the fact that these different processes have distinct and non-overlapping time constants which characterize their decays. The experimental definitions of these processes are as follows. Facilitation refers to the short term process as characterized by Mallart & Martin (1967). Facilitation typically decays with a double exponential time course with time constants of about 50 and 300 msec. Augmentation, which is the topic of this paper, has a time constant of about 7 sec. Augmentation thus has a time course intermediate in duration between facilitation and potentiation. Potentition refers to the long term process as defined by Magleby & Zengel (1975a, b). Potentiation typically decays with a time constant of about 20 see following a few impulses. The time constant can increase to minutes following hundreds to thousands of impulses. Potentiation is thought to represent the same process as post-tetanic potentiation (Magleby, 1973b) previously studied by Liley (1956), Gage & Hubbard (1966), and Rosenthal (1969). From these definitions it can be seen that about 150 msec will be required for the faster component of facilitation to reach a steady state (within 5 %) during repetitive stimulation or to decay to insignificant levels following repetitive stimulation. These same conditions will be met in about 1000 msec for the slow component of facilitation, in about 20 see for augmentation, and in 60-100's of sec for potentiation. RESULTS

Augmentation decays with a time constant of about 7 sec The phenomenon under investigation is illustrated in Fig. 1 where an example of experimental data and the derivation of augmentation from these data are presented. Fig. 1 A presents a plot ofe.p.p. amplitudes against time obtained from the average of ten identical conditioning-testing trials in which e.p.p.s were recorded with a surface electrode. For each conditioning-testing trial the nerve was first stimulated once every 5 sec to establish a control response. The nerve terminal was then conditioned by A

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455 AUGMENTATION OF TRANSMITTER RELEASE stimulating 300 times at 20/sec, and the effect of this conditioning stimulation was followed by testing once every 1-5 sec for 6 impulses and then once every 5 sec for 59 impulses. (Fig. 1 B presents a computer-sampled e.p.p. recorded during one of the individual trials. The horizontal lines 10

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Fig. 1. Effect of repetitive stimulation on augmentation and potentiation. A, surface recorded e.p.p. amplitudes before, during, and following 300 conditioning impulses at 20/sec. The nerve was first stimulated once every 5 sec to establish a control response. Following the conditioning train the nerve was tested once every 1-5 sec for 6 impulses and then once every 5 see for 59 impulses. Data averaged from 10 identical trials. B, computer sampled e.p.p. recorded during the conditioning train from one of the individual trials averaged in A. The difference between the horizontal lines is taken as the e.p.p. amplitude (see methods). C, decay of augmentation, A(t), and potentiation, P(t), plotted semilogarithmically as a function of time after the conditioning stimulation. Same data as in A. The filled circles represent the decay of V(t), the fractional increase in e.p.p. amplitude (eqn. (4)); the line through the filled circles, determined by a least squares fit to data points beyond 30 sec, represents the exponential decay of P(t), which had a time constant, Tp, of 60 sec. P(T), the initial magnitude of potentiation immediately following the conditioning train, is given by the intercept of this line with the ordinate at 0 time and was 1-3. Estimates of augmentation,A(t),were obtained asuming a multiplicative (open circles) or additive (filled squares) relationship with P(t) by dividing or subtracting off the effect of P(t) from V(t) as described by eqns. (5) and (6). The lines through the open circles and filled squares represent least squares fits to the first 15 see of the decays. A(T), the initial magnitude of augmentation immediately following the conditioning train, is given by the intercept of these lines with the ordinate at 0 time, and was 1 1 assuming a multiplicative relationship with P(t) or 2-6 assuming an additive relationship. The corresponding time constant for the decay of augmentation, TA' was 8-3 (multiplicative) or 7-7 sec (additive).

K. L. MAGLEBY AND JANET E. ZENGEL 456 represent the computer-determined base line and peak e.p.p. amplitude. The vertical distance between these lines is taken as the e.p.p. amplitude.) The first ten points in Fig. 1 A represent the control responses. During the conditioning train the e.p.p. amplitudes rapidly increase to over 14 times this control level, and then decay back to the control level in the posttetanic period with at least three apparent time constants. The immediate 60 % drop in response following the conditioning train is due mainly to the rapid decay of facilitation which has a time course of less than 1 sec (Mallart & Martin, 1967; Magleby, 1973a, b). After the rapid decay of facilitation the e.p.p. amplitudes then decay with what appears to be two time constants. When this decay, expressed as the fractional increase in e.p.p. amplitude (V(t) in eqn. (4)), is plotted semilogarithmically against time in Fig. 1C (filled circles), a dual exponential decay becomes readily apparent. The continuous line through the filled circles in Fig. 1C, determined by a least squares fit to the data points beyond 30 sec, represents the exponential decay of potentiation which had a time constant of 60 sec in this experiment. The intercept of this line with the ordinate at 0 time is taken as the magnitude of potentiation immediately following the conditioning train and was 1P33. Superimposed on the slow decay of potentiation is a faster decaying phenomenon which will be referred to as augmentation. Whether augmentation is actually different from facilitation and potentiation and what role it plays in transmitter release are examined in this and the following paper (Magleby & Zengel, 1976). It is not known how augmentation and potentiation interact. The open circles represent the decay of augmentation assuming a multiplicative relationship between augmentation and potentiation, such that V(t) = (A(t) + 1) (P(t) + 1)- 1, (5) where V(t) is the total fractional change in e.p.p. amplitude compared to the control as defined by eqn. (4), A(t) is augmentation, and P(t) is potentiation. The filled squares represent the decay of augmentation assuming an additive relationship such that V(t) = A(t)+P(t). (6) Notice that facilitation must be 0 for eqns (5) and (6), which is the case at times greater than 1-2 sec after the conditioning train when these equations are used to derive augmentation. From Fig. 1 C, it is apparent that augmentation decays approximately exponentially over at least two time constant units. The decay of augmentation, then, may be approximated by A(t)

=

A(TA) e'T.A

(7)

AUGMENTATION OF TRANSMITTER RELEASE 457 where A(t) is augmentation t sec after the end of the conditioning train, A(T) is the initial magnitude of augmentation immediately following T sec of conditioning stimulation, and rA is the time constant characterizing the time course of decay. A(T) is determined experimentally by projecting the exponential decay of A(t) back to intercept the ordinate at 0 time (the end of the conditioning train) and rA is taken as the time required for A(t) to decay to 1/e of its initial value. For the experiment in Fig. 1, the magnitude of augmentation immediately following the conditioning stimulation, A(T) in eqn. (7), was 1-12 assuming a multiplicative relationship between potentiation and augmentation and 2-61 assuming an additive relationship. The corresponding time constant for decay, T din eqn. (7), was 8-3 see assuming a multiplicative relationship and 7-7 sec assuming an additive relationship. The decay of augmentation usually deviated slightly from a simple exponential decay, with augmentation decaying faster immediately after the conditioning train. This deviation was usually barely perceptible when the initial magnitude of augmentation, A(T), was less than about 0.5 (assuming a multiplicative relationship between A(t) and P(t)) and increased as the magnitude of augmentation increased. Some possible explanations for this deviation will be discussed in the following paper (Magleby & Zengel, 1976). It should be pointed out that the few rapid testing impulses applied immediately after the conditioning train to test for augmentation do not significantly alter estimates of its magnitude or time course. In a series of experiments similar to those shown in Fig. 1, changing the number of the rapid testing impulses in the range of 1-6 had little or no effect on estimates of augmentation or potentiation. It should also be mentioned that estimates of potentiation appeared similar whether the testing impulses used to follow the decay of potentiation were applied once every 5 sec or once every 10 sec. The magnitude of augmentation increases with the number of conditioning impulses while the time constant remains relatively unchanged Representative plots of the magnitudes and time constants for the decay of augmentation observed in thirty-three experiments are presented in Fig. 2 where estimates of these parameters are plotted against the number of conditioning impulses delivered at 20/sec. These estimates were derived by the method shown in Fig. 1, assuming a multiplicative relationship between A(t) and P(t) for Fig. 2A and B and an additive relationship for Fig. 2C and D. These Figures show that the magnitude of augmentation, A(T), tends to increase with the number of the conditioning impulses. A(T) ranged from 0-3 to 0 6 following 50 impulses to 0-5 to 7-8 following

K. L. MAGLEBY AND JANET E. ZENGEL 458 600 impulses assuming a multiplicative relationship between A(t) and P(t). A(T) increased to over 15 assuming an additive relationship. Because the data in Fig. 2 were obtained from a large number of preparations with only one to three durations of conditioning stimulation used in each preparation, the data must be viewed only as a general representation of the effect of increasing the duration of stimulation on the plotted parameters. Within any single preparation estimates of A(T) always increased monotonically with the duration of stimulation. In contrast to the increase in the magnitude, the time constant for the decay of augmentation, TA, remained relatively unchanged as the number of conditioning impulses increased, with a mean value of 7-2 sec (multiplicative, dashed line in Fig. 2B) or 6-9 sec (additive, dashed line in Fig. 2D). The time constants, TA, in Fig. 2 were usually estimated from about the first 20 sec of decay of augmentation after the conditioning train. If TA were estimated from the first 10 sec of decay there was a tendency for TA to become shorter as the duration of stimulation was increased, because as A(T) increased with the duration of stimulation, the decay of augmentation deviated increasingly more from a simple exponential, decaying faster immediately after the conditioning train. The most striking feature of augmentation shown in Fig. 2 is the wide range of magnitudes that were observed following identical conditioning trains. This variation was not due to measurement errors, for repeatable estimates of A(T) were obtained from several consecutive trials on the same preparation. The variation appeared to arise for two reasons: actual differences between preparations and long term changes in the properties of the preparations that occurred during the 4-16 hr experiments. The open circles in Fig. 2 represent estimates of A(T) and TA made from data *

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459 AUGMENTATION OF TRANSMITTER RELEASE obtained early in an experiment. The filled circles are representative estimates that were obtained from data collected later in the experiment or from data averaged from an entire experiment. It can be seen that the magnitude of augmentation tends to be less for data collected early in an experiment. A detailed analysis of this effect and some possible explanations for the large variations in A(T) following identical conditioning 16

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Fig. 2. Effect of the number of conditioning impulses delivered at 20sec on augmentation and potentiation. Open circles: data obtained early in an experiment. Filled circles: data obtained from later in the experiment or averaged from an entire experiment. Data from thirty-three preparations. A-D, estimates of the magnitude, A (T), and time constant, TA, of augmentation plotted as a function of the number of conditioning impulses. These estimates were obtained assuming a multiplicative (A, B) or additive (C, D) relationship with P(t). The dashed lines in B and D represent the mean values of TA for these data. E, F, estimates of the magnitude, P(T), and time constant, Tp, of potentiation plotted as a function of the number of conditioning impulses. Some points have been moved laterally for clarity.

K. L. MAGLEBY AND JANET E. ZENGEL 460 trains will be presented in the following paper (Magleby & Zengel, 1976). The important point for now is that a systematic variation in augmentation occurs. A quantitative analysis of augmentation must await an explanation for this variation. Fig. 2E and F shows the corresponding estimates of the parameters characterizing potentiation for the same preparations that were presented in Fig. 2A-D. The magnitude, P(T), and time constant, rp, for the decay of potentiation are seen to increase with the duration of conditioning stimulation as has been previously reported (Magleby & Zengel, 1975a, b). These data are presented to show (1) that potentiation always decayed at least 3-4 times slower and typically 10 times slower than augmentation (the time constants of these two processes were never observed to overlap) and (2) that the magnitude of augmentation can be considerably greater than the magnitude of potentiation. The maximum observed magnitude for P(T) in these experiments was about 2 for 600 impulses at 20/sec. Corresponding estimates of A(T) could be greater than 7 or 16 depending on whether A(t) is multiplicative or additive to P(t). Notice in Figs. 1 and 2 that estimates of augmentation derived assuming an additive relationship with potentiation have a 1-3 times greater magnitude and a slightly shorter time course than those derived assuming a multiplicative relationship. To simplify discussion, estimates of augmentation presented in the rest of this paper will refer to those derived assuming a multiplicative relationship (eqn. (5)) unless indicated. This simplification seems justified for now since qualitatively similar results were obtained when the data were analysed assuming an additive relationship. A true multiplicative relationship between augmentation and potentiation is consistent with the idea that the joint probability of these processes determines transmitter release (see Magleby, 1973b). Although it is suggested in the following paper (Magleby & Zengel, 1976) that these processes may be different, we do not exclude the possibility that augmentation and potentiation have an additive relationship.

Augmentation, facilitation, and potentiation all result from an increase in the number of quanta released from the nerve terminal The necessary first step in characterizing augmentation is to determine whether it is a pre- or a post-synaptic phenomenon. Although it seems unlikely that augmentation arises from a transient increase in postsynaptic sensitivity to acetylcholine, this possibility was examined by using spontaneously released quantal packets of acetylcholine to test for possible changes in post-synaptic sensitivity during the rise and decay of augmentation. Since the mean size of the quantal packets of acetylcholine

461 AUGMENTATION OF TRANSMITTER RELEASE released from the nerve terminal remains constant (except under conditions of drug treatment or extremely prolonged stimulation, neither of which was used in this paper) any change in mean m.e.p.p. amplitude would indicate a change in post-synaptic sensitivity (del Castillo & Katz, 1954a; Elmqvist & Quastel, 1965a; Jones & Kwanbunbumpen, 1970). An example of an experiment in which miniature end-plate potential (m.e.p.p.) amplitudes were recorded before, during and following repetitive stimulation to test for changes in post-synaptic sensitivity is shown in Fig. 3. The continuous line in Fig. 3A presents the mean e.p.p. amplitude (normalized in terms of m.e.p.p. amplitude) plotted against time for the average of 11 intracellularly recorded conditioning-testing trains similar to that shown in Fig. 1 A, except that the conditioning stimulation (indicated by the horizontal bar) was 400 impulses at 20/sec. In this preparation, the average e.p.p. amplitude increased more than 6 times during the conditioning stimulation and then returned to control. Fig. 3B-F presents m.e.p.p.s and e.p.p.s recorded intracellularly during one of the eleven conditioning-testing trials used for the averaged data presented in Fig. 3A. The brackets in Fig. 3A indicate at what points in time the data shown in Fig. 3B-F were taken. From these data it appears that the amplitude of the m.e.p.p.s (indicated by arrows) did not change during or immediately following the conditioning-testing train. This is more clearly shown in Fig. 3 G which presents the mean + S.D. of the amplitudes of all the m.e.p.p.s occurring during two of the eleven trials used for the average data. Each data point is placed in the middle of the time interval from which the m.e.p.p.s for that point were averaged. If augmentation were due to an increased post-synaptic sensitivity, then to account for the magnitude of augmentation which was greater than 1 in this experiment, m.e.p.p. amplitudes would have to be greater than twice their control amplitude immediately following the conditioning stimulation. However, since the m.e.p.p. amplitudes remained unchanged during and following the conditioning stimulation in this and two additional experiments, including one in which the magnitude of augmentation was greater than 3, it can be concluded that augmentation as well as facilitation (del Castillo & Katz, 1954b) and potentiation (Hutter, 1952; Liley, 1956; Elmqvist & Quastel, 1965b; Weinreich, 1971) are of presynaptic origin and do not result from changes in post-synaptic sensitivity. Thus, these three processes are all expressed as an increase in transmitter release. Facilitation has been shown to result from an increase in the number of quantal packets of acetylcholine released from the nerve terminal, as opposed to an increase in the size of these quantal packets (del Castillo & Katz, 1954b; Liley, 1956; Dudel & Kuffler, 1961). It is generally concluded that potentiation also results from an increase in the number of quanta

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463 AUGMENTATION OF TRANSMITTER RELEASE released (Liley, 1956; Maeno & Edwards, 1969; Hubbard et al. 1971; Bennett, Florin & Hall, 1975). It seems reasonable, then, to assume that augmentation, like facilitation and potentiation, results from an increase in the number of quanta released. However, a necessary second step in characterizing augmentation is to determine if augmentation does result from an increase in the number of quanta released as opposed to an increase in quantal size. This was done by examining the statistics of quantal

transmitter release. From the data in Fig. 3B-F it can be seen that the probability of a failure of response is greatest during the control periods (B and F), and decreases during and immediately following the conditioning train (C, D, and E), suggesting that the probability of quantal release is increased during and following repetitive stimulation. If augmentation results from an increase in the number of quantal packets of acetylcholine released per impulse (as suggested from Fig. 3 B-F), then estimates of quantal content, m, obtained from mean e.p.p. amplitude mean m.e.p.p. amplitude should equal estimates obtained from number of nerve impulses m = loge number of failures of e.p.p. response (del Castillo & Katz, 1954a; Boyd & Martin, 1956; Liley, 1956). This is found to be the case as shown in Fig. 3A which presents estimates of quantal content obtained before, during, and following repetitive stimulation by these two independent methods. Each data point before and after the conditioning train represents the mean quantal content of ten consecutive responses from each of the eleven trials. The data points during the train represent the mean quantal contents of eighty consecutive responses from each of the eleven trials. If augmentation resulted from an increase in the size of the quantal packets of acetylcholine instead of from an increase in the number released, the two different estimates of quantal content immediately after the conditioning train would have to differ by more than a factor of 2 to account for the magnitude of augmentation at this time. This is not the case. Estimates of quantal content obtained by the two methods are found to superimpose immediately after the condidioning train, when augmentation is known to be present, as well as during the train. Since facilitation and potentiation also contribute to the increase in e.p.p. amplitudes during the conditioning train, the excellent agreement between the two independent estimates of quantal content before, during, and following the conditioning train indicates that facilitation,

464 K. L. MAGLEBY AND JANET E. ZENGEL augmentation, and potentiation all result from an increase in the number of quantal packets of acetylcholine released from the nerve terminal by each impulse. The time constant for the decay of augmentation ha7 a Q1o near 4 In order to determine whether the decay of augmentation results from a passive process such as a simple diffusion of some residual substance in the nerve terminal, or from a more complicated process such as the active removal of this substance, we have examined the effect of temperature on the time constant for the decay of augmentation. A similar study has also been made on potentiation in order to compare the Q10 for the decays of the two processes. The ideal way to measure the Q10 for the decay of these two processes would be to measure the decay of each process following a single conditioning impulse delivered at a series of different temperatures. Experimentally this is not possible since neither process is large enough to measure after one impulse. Several hundred conditioning impulses are usually required for an accurate determination of the time constants. This raises a difficult problem since the time constants for the decays of potentiation (Magleby & Zengel, 1975a) and augmentation (K. L. Magleby & J. E. Zengel, unpublished observations) often appear to be functions of the frequency of the conditioning stimulation. If, for example, a process has a Q10 of 4, raising the temperature from 10 to 200 C will lead 5

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AUGMENTATION OF TRANSMITTER RELEASE 465 to a 4 times increase in the rate at which the process decays. If the stimulation rate is kept constant during this temperature increase, the nerve terminal will respond as if the stimulation rate were really 1/4 the delivered rate since the relevant processes in the nerve terminal now operate 4 times faster but the stimulation rate has remained the same. Thus, if the decay of a process is a function of the stimulation rate, estimates of the Q10 of this decay determined by changing the temperature while keeping the stimulation rate constant can be in error. The solution to the problem would be to change the stimulation rate to correspond to the Q10 of the process and the experimental temperature change. For example, if a process has a Q10 of 4 and the experimental temperature change is 50 C, 5

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466 K. L. MAGLEBY AND JANET E. ZENGEL the stimulation rate should be twice as fast at the higher temperature so that the relationship between the stimulation rate and the rate at which the nerve terminal operates remains constant. It thus appears that it is only possible to obtain an exact determination of the Q10 for the decay of augmentation or potentiation if the Q10 is already known. To approach this problem estimates of Q10 were made both by keeping the stimulation rate constant (assumed Q10 of 1) and by increasing the stimulation rate to correspond to an assumed Q10 of 3 or 4. If the true Q10 falls in the range of 1-4 (a reasonable assumption) then the range of experimental determinations of Q10 obtained in this manner should include the true Q1o. An example of the effect of temperature on the decay of augmentation and potentiation for one preparation is shown in Fig. 4, which presents semilogarithmic plots of the decay of e.p.p. amplitudes following conditioning stimulation at 200 C (Fig. 4A) and at 150 C (Fig. 4B). For the 200 C data the nerve terminal was stimulated with single impulses at once per 5 sec, then conditioned with 600 impulses at 20/sec, followed by 6 testing impulses at once per 1-5 sec and 80 impulses at once per 5 sec. For the 15° C data a Q10 of 4 was assumed so the stimulation rates for the control, conditioning, and testing trains were half the 200 C rates. It can be seen that augmentation and potentiation decay slower at the lower temperature. The time constant of decay of augmentation increased from 10-3 sec at 200 C to 21-6 sec at 150 C for a Q10 of 4.4, and the time constant of decay of potentiation increased from 61 to 94 sec for a Q10 of 2-3. Fig. 5A presents estimates of the Q10 for the decay of augmentation plotted against the temperature range over which these estimates were determined for a series of experiments similar to that shown in Fig. 4. These estimates were made either by keeping the stimulation rate the same at the two temperatures (assumed Q10 of 1, continuous lines indicating the temperature range) or by stimulating faster at the higher temperatures (assumed Q10 of either 3 or 4, dashed lines). The mean Q10 for the decay of augmentation from these determinations was 3-8 + 1-7 (S.D.) suggesting that the decay of augmentation is not due to simple passive diffusion of some substance through an aqueous solution away from its site of action, but is determined by a more complicated process. The average Q10 of potentiation determined in a similar manner was found to be 2*4 + 0-8 as shown in Fig. 5B, suggesting that the decay of potentiation is also not due to simple diffusion. This value is in agreement with the value of 2*5 found by Rosenthal (1969) for the decay of potentiation under conditions of much higher transmitter release. The Q10 of 3-8 for the time constant of decay of augmentation is closer to the Q10 of about 4 for the decay of facilitation (Eccles, Katz & Kuffler, 1941; Balnave & Gage, 1974) than it is to the Q10 of 2-4 for the decay of potentiation.

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DISCUSSION

This paper examines the properties of a previously uncharacterized process that acts to increase e.p.p. amplitudes following repetitive stimulation. We have used the non-committal name augmentation for this process since it is not known whether it is a slow facilitation, a fast potentiation, or a new process. If it is later shown that augmentation increases e.p.p. amplitudes by the same mechanism as either facilitation or potentiation then the name can be appropriately changed to reflect this association. Augmentation was found to decay approximately exponentially with a mean time constant of decay of about 7 sec. Thus augmentation has a time course intermediate in duration between facilitation, which decays in less than 1 sec (Mallart & Martin, 1967; Magleby, 1973a, b), and potentiation, which decays with a time constant of tens of seconds to minutes (Rosenthal, 1969; Magleby & Zengel, 1975a). The marked

K. L. MAGLEBY AND JANET E. ZENGEL differences (typically tenfold) in time constants between these three processes is consistent with the hypothesis that different mechanisms determine their decays. The magnitude of augmentation increased with the duration of the conditioning stimulation while the time constant remained relatively unchanged. The magnitude ranged from 03-0-6 following 50 impulses at 20/sec to 0*5-7*8 following 600 impulses at 20/sec (assuming a multiplicative relationship between augmentation and potentiation). (By definition, eqn. (2), an augmentation of 0 3 increases e.p.p. amplitude 1*3 times.) The important points to be drawn from these observations are (1) that augmentation is usually present following repetitive stimulation, and (2) that the magnitude of augmentation is often sufficiently great to become a major factor in increasing e.p.p. amplitudes following (and presumably during) repetitive stimulation. Depending on the experiment, the magnitude of augmentation can be significantly greater than both potentiation, which in this study reached magnitudes of 1-2 following 600 impulses at 20/sec, and facilitation, which reaches a steady-state level of about 1-2 for this stimulation rate (Mallart & Martin, 1967). In view of the fact that augmentation has gone relatively unnoticed, we were quite surprised to find that it is usually present following repetitive stimulation, and that it can reach such large magnitudes. The question arises then whether augmentation is a general characteristic of neuromuscular and synaptic transmission or whether it is generated by our experimental technique. Three lines of evidence argue that augmentation is a characteristic of synaptic transmission. Firstly, an apparent augmentation following stimulation is also evident in the data of Larrabee & Bronk (1947) from the cat sympathetic ganglia, in the data of Liley (1956) from the rat diaphragm, and in the data of Landau et al. (1973) from the frog sartorius. Secondly, we have obtained similar results on augmentation in both frogs and toads. Thirdly, augmentation is present when single testing impulses are placed at a variable interval following each of a series of conditioning trains (Magleby & Zengel, 1975a), suggesting that the few rapid testing impulses that are used in the present study to detect augmentation before it decays do not generate augmentation. It was found in this paper that, like facilitation (del Castillo & Katz, 1954b; Liley, 1956; Dudel & Kuffler, 1961) and potentiation (Liley, 1956; Maeno & Edwards, 1969; Bennett et al. 1975), augmentation is a presynaptic phenomenon, arising from an increase in the number of quantal packets of acetylcholine released from the nerve terminal by each impulse. It was also found that the time constant of decay of augmentation has a Q10 of about 4, suggesting that the decay of augmentation is not due to simple passive diffusion through an aqueous solution, but is dependent on 468

469 AUGMENTATION OF TRANSMITTER RELEASE a more complicated process. There are many factors involved in transmitter release, as reviewed by Hubbard (1973), and augmentation could reflect a change in one or more of these factors. Which factors are involved, however, is unknown. Estimates of the magnitude of potentiation in this study usually tended to be less than in our previous work (Magleby & Zengel, 1975a, b) and estimates of the time constant for the decay of potentiation usually tended to be longer. These differences most likely occur because in our previous work we probably underestimated the contribution of augmentation to the decays of e.p.p. amplitudes after repetitive stimulation and because in the present study data were collected for longer periods of time which can lead to this type of change in potentiation (Magleby & Zengel, 1976). In summary, augmentation can be an important factor in increasing transmitter release and will therefore have to be accounted for when using repetitive stimulation to study the function of the nerve terminal or when formulating models of transmitter release. Supported by USPHS Grant NS10277. REFERENCES BALNAVE, R. J. & GAGE, P. W. (1974). On facilitation of transmitter release at the toad neuromuscular junction. J. Physiol. 239, 657-675. BENNEr, M. R., FLORIN, T. & HAmT, R. (1975). The effect of calcium ions on the binomial statistic parameters which control acetylcholine release at synapses in striated muscle. J. Phy8iol. 247, 429-446. BOYD, I. A. & MARTIN, A. R. (1956). The end-plate potential in mammalian muscle. J. Phy8iol. 132, 74-91. BRAuN, M., SCHMIDT, R. F. & ZIxm ERmANN, M. (1966). Facilitation at the frog neuromuscular junction during and after repetitive stimulation. Pfluger8 Arch. ge8. Phyeiol. 287, 41-55. DEL CASTILLO, J. & KATZ, B. (1954a). Quantal components of the end-plate potential. J. Phywiol. 124, 560-573. DEL CASTILLO, J. & KATZ, B. (1954b). Statistical factors involved in neuromuscular facilitation and depression. J. Physiol. 124, 574-585. DuDEL, J. & KuFFLER, S. W. (1961). Mechanism of facilitation at the crayfish neuromuscular junction. J. Phyiol. 155, 530-542. ECCLES, J. C., KATZ, B. & KUFFLER, S. W. (1941). Nature of the 'endplate potential' in curarized muscle. J. Neurophyeiol. 4, 362-387. ELMQVIST, D. & QUASTEL, D. M. J. (1965a). Presynaptic inhibition of hemicholinium at the neuromuscular junction. J. Physiol. 177, 463-482. ELMQVIST, D. & QUASTEL, D. M. J. (1965b). A quantitative study of end-plate potentials in isolated human muscle. J. Phy&iol. 178, 505-529. FENG, T. P. (1941). Studies on the neuromuscular junction. XXVI. The changes of the end-plate potential during and after prolonged stimulation. Chin. J. Phywiol. 16, 341-372.

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GAGE, P. W. & HUBBARD, J. I. (1966). An investigation of the post-tetanic potentiation of end-plate potentials at a mammalian neuromuscular junction. J. Physiol. 184, 353-375. HUBBARD, J. I. (1963). Repetitive stimulation at the neuromuscular junction, and the mobilization of transmitter. J. Physiol. 169, 641-662. HUBBARD, J. I. (1973). Microphysiology of vertebrate neuromuscular transmission. Phy8iol. Rev. 53, 674-723. HUBBARD, J. I., JONES, S. F. & LANDAU, E. M. (1971). The effect of temperature change upon transmitter release, facilitation, and post-tetanic potentiation. J. Physiol. 216, 591-609. HUTTER, 0. F. (1952). Post-tetanic restoration of neuromuscular transmission blocked by d-tubocurarine. J. Physiol. 118, 216-227. JONES, S. F. & KWANBUNBUMPEN, S. (1970). Some effects of nerve stimulation and hemicholinium on quantal transmitter release at the mammalian neuromuscular junction. J. Physiol. 207, 51-61. LANDAU, E. M., SMOLINSKY, A. & LAss, Y. (1973). Post-tetanic potentiation and facilitation do not share a common calcium-dependent mechanism. Nature, New Bigo. 244, 155-157. LARRABEE, M. G. & BRONK, D. W. (1947). Prolonged facilitation of synaptic excitation in sympathetic ganglia. J. Neurophy8-iol. 10, 137-154. Liixy, A. W. (1956). The quantal components of the mammalian end-plate potential. J. Physiol. 133, 571-587. MAENO, T. & EDWARDS, C. (1969). Neuromuscular facilitation and low-frequency stimulation and effects of some drugs. J. Neurophysiol. 32, 785-792. MAGLEBY, K. L. (1973a). The effect of repetitive stimulation on facilitation of transmitter release at the frog neuromuscular junction. J. Physiol. 234, 327-352. MAGLEBY, K. L. (1973b). The effect of tetanic and post-tetanic potentiation on facilitation of transmitter release at the frog neuromuscular junction. J. Physiol. 234, 353-371. MAGLEBY, K. L. & ZENGEL, J. E. (1975a). A dual effect of repetitive stimulation on post-tetanic potentiation of transmitter release at the frog neuromuscular junction. J. Physiol. 245, 163-182. MAGLEBY, K. L. & ZENGEL, J. E. (1975b). A quantitative description of tetanic and post-tetanic potentiation of transmitter release at the frog neuromuscular junction. J. Physiol. 245, 183-208. MAGLEBY, K. L. & ZENGEL, J. E. (1976). Long term changes in augmentation, potentiation, and depression of transmitter release as a function of repeated synaptic activity at the frog neuromuscular junction. J. Physiol. 257, 471-494. MALRT, A. & MARTIN, A. R. (1967). An analysis of facilitation of transmitter release at the neuromuscular junction of the frog. J. Physiol. 193, 679-694. MARTIN, A. R. (1955). A further study of the statistical composition of the end-plate potential. J. Physiol. 130, 114-122. ROSENTHAL, J. (1969). Post-tetanic potentiation at the neuromuscular junction of the frog. J. Physpol. 203, 121-133. TRIEs, R. E. (1965). Neuromuscular depression and the apparent depletion of transmitter in mammalian muscle. J. Neurophysiol. 28, 427-442. WEINREICH, D. (1971). Ionic mechanism of post-tetanic potentiation at the neuromuscular junction of the frog. J. Physiol. 212, 431-446. ZENGEL, J. E. & MAGLEBY, K. L. (1975). A possible new process that acts to increase transmitter release at the frog neuromuscular junction. Biophys. J. 15, 127a.