J. Physiol. (1975), 246, pp. 109-142 With 20 text-ftgure8

109

Printed in Great Britain

LITHIUM IONS AND THE RELEASE OF TRANSMITTER AT THE FROG NEUROMUSCULAR JUNCTION

By A. C. CRAWFORD From the Physiological Laboratory, Downing Street, Cambridge, CB2 3EG

(Received 10 May 1974) SUMMARY

1. Transmitter release has been examined at the frog neuromuscular junction when all or part of the Na of the Ringer is replaced by Li ions. 2. No immediate change occurs in either the mean quantal content of the end-plate potential or the miniature end-plate potential frequency on changing to Li Ringer but over the following hour both these quantities increase by more than two orders of magnitude. 3. During the first 30-40 min of an exposure to Li-Ringer the m.e.p.p. frequency rises exponentially with a time constant of 10 min, and the mean quantal content of the e.p.p. grows by addition of extra evoked quanta, the increment rising exponentially with a time constant the same as that of the m.e.p.p. frequency. 4. Following this initial period in Li-Ringer the m.e.p.p. frequency accelerates to a peak of several hundred quanta per second and then declines slowly over the next few hours. Just before the m.e.p.p. frequency peak the conduction velocity of the presynaptic action potential declines and shortly afterwards synaptic transmission fails as the action potential no longer conducts into the terminals. 5. The rise in the m.e.p.p. frequency during the first 30-40 min is independent of the [Ca2+]o. At subsequent times before the peak external Ca becomes progressively more effective in accelerating the m.e.p.p. frequency and in the presence of 1 mM-EGTA spontaneous release stabilizes at 60-80 quanta/sec. 6. The [Li+]o strongly influences the rate of increases in both evoked and spontaneous release but not their extent; replacing only half the Na of the Ringer by Li increases the time constants of the increases to about 30 min. 7. Rises in the m.e.p.p. frequency can be irreversibly accelerated by tetanizing the nerve in a Li-Ringer in which the Ca has been chelated by EGTA. The extent of the increase in the m.e.p.p. frequency is dependent

A. C. CRAWFORD on the number of pulses in the tetanus and is little affected by the frequency of stimulation. Accumulation of Li ions inside the presynaptic terminals probably underlies the changes in spontaneous release. 8. When only 10 % of the Na of the Ringer is replaced by Li+ ions the magnitude of post-tetanic potentiation of the e.p.p. and of the posttetanic rise in the m.e.p.p. frequency is increased. Under these conditions changes in facilitation of the e.p.p. are small. 9. Various mechanisms by which Li could alter transmitter release are discussed and it is suggested that intracellular Ca sequestering mechanisms of the presynaptic terminals are affected when an end-plate is exposed to Li-Ringer. 110

INTRODUCTION

Since Overton (1902) first showed that muscle retained its excitability in Li solutions there has been much interest devoted to the effects of Li+ on the electrical properties of excitable cells. The action potential is little affected when the Na of the external solution is replaced by Li (Hodgkin & Katz, 1949; Huxley & Stimpfli, 1951) since the Na channel of the action potential discriminates poorly between the two alkali metal cations (Chandler & Meves, 1965). Fatt (1950) demonstrated that acetylcholine applied to muscle fibres still produced a depolarization in Li-Ringer and Onodera & Yamakawa (1966) were able to record end-plate potentials and miniature end-plate potentials at frog neuromuscular junctions when all of the Na of the Ringer had been replaced by Li. The effect of Li-Ringer on transmitter release has been studied by many authors with conflicting results. If part of the sodium of the Ringer is replaced isotonically with sucrose or glycine small increases in both spontaneous and evoked transmitter release occur (Birks & Cohen, 1965; Kelly, 1965, 1968; Gage & Quastel, 1966; Colombo & Rahamimoff, 1968), but substitution of Na by either Li or tetramethylammonium ions fails to produce this effect (Onodera & Yamakawa, 1966; Kelly, 1968). Indeed Li solutions have been reported to block synaptic transmission irreversibly (Kelly, 1968) and to produce high frequency bursts of miniature end-plate potentials (Onodera & Yamakawa, 1966; Kelly, 1968), both effects being attributed to depolarization of the nerve terminals. In this paper large increases in the mean quantal content of the endplate potential and miniature end-plate potential frequency are reported, that develop slowly when neuromuscular junctions are exposed to Li+ ions over periods of hours rather than minutes. Evidence is presented that the increases are dependent on Li entry into the presynaptic terminals. The effects are most easily explained by an action on the Ca sequestering systems of the presynapse.

TRANSMITTER RELEASE AND Li+

III

METHODS Experiments were carried out on frog (Rana temporaria) sartorius-sciatic preparations at room temperature (18-24° C). Standard intracellular recording techniques with 3M-KCl-filled micro-electrodes of tip resistance 10-20 MC were used to record from superficial end-plates of the muscle. The bathing solution was changed by flowing 10-20 times the bath volume (about 3 ml.) over the preparation whilst the end-plate was impaled by the micro-electrode. For focal recording from terminals within an end-plate the preparation was mounted in a chamber similar to that used by McMahan, Spitzer & Peper (1972) and viewed with a Ziess x 40 water immersion lens at x 400 magnification. Both bright field and phase contrast optics were available. In locating terminals it proved quite easy to see the nuclei of the overlying Schwann cells and most recordings were carried out at spots just beyond the Schwann cell nucleus in the long axis of the muscle fibre. Electrodes for focal recording were filled with either 0-5 M-NaCl or 0-5 M-LiCl depending on the major cation of the Ringer, and were advanced at an angle of about 300 from the horizontal, under the water immersion lens.

Solutiomw Table 1 shows the composition of the main solutions used in these experiments. All solutions contained neostigmine methylsulphate 10-6 g/ml. Columns A and B show the basic Na+ and Li+ Ringer used for following changes in the evoked and spontaneous release of transmitter. LiCl was obtained from the Fischer Chemical Co, Fair Lawn, New Jersey. In all solutions neutrality (pH 7.2) was obtained by addition of adequate amounts of NaOH (B.D.H.) or LUOH (Sigma) as appropriate. TABLE 1. Composition of solutions E F C B D Ion A Na 114-5 97-5 Li 97.5 97.5 118-5 114-5 20-0 K 2-0 2-0 2-0 2-0 2-0 3-2 3-2 3-2 3-2 3-2 3-2 Mg 0-4 Ca 0-4 Tris 2-5 2-5 22-5 22-5 2-5 2-5 1.0 1-0 1-0 1-0 EGTA 126-2 126-2 125-4 Cl 125-4 125-4 31-9 Glucuronate Gluconate 94.5 Note: all solutions contain neostigmine methylsulphate, 10-6 g/ml. All ion concentrations are in gm-ion/I.

Solution C is a 'Ca-free' Li-Ringer in which no Ca has been added and the free Ca concentration reduced to a calculated value of less than 10-9 M by the addition of 1 mM Li-EGTA (Sillen & Martell, 1964; Miledi & Thies, 1971). Solutions D to F were used in experiments where the preparation was depolarized with raised external K+ ions (20 mm K+). They are essentially Cl-free Ringer containing large monovalent organic anions as the substitute. All contain the same concentration of Na+ or Li+, changes in [K+] being achieved by isotonic replacement by Tris-glucuronate. In solution E the counter-ion to Na was gluconate (Fisons Chemicals Ltd). All other Cl replacements were for glucuronate (Sigma). Salts of glucuronic acid were produced

112

A. C. CRAWFORD

by titration to pH 7-2 of the appropriate base (as the hydroxide). Chloride substitution was necessary to increase the input resistance of the muscle fibre and hence the miniature end-plate potential amplitude. In Li-Ringer the quantal size is about halved (Onodera & Yamakawa, 1966); in EGTA-Ringer the electrode seals into the muscle fibre poorly (Hubbard, Jones & Landau, 1968 a) and K depolarization reduces the driving potential for transmitter action (Takeuchi & Takeuchi, 1960). These effects combine to make the m.e.p.p. amplitude too small to be followed in Cl-based Ringer. Since the C1 conductance contributes significantly to the muscle fibre input resistance at pH 7-2 (Hutter & Noble, 1960; Hutter & Warner, 1967) the substitution of Cl by an impermeant anion can significantly raise the miniature end-plate potential (m.e.p.p.) amplitude. Complete substitution of the sodium of a Ringer by Li+ ions reduces the m.e.pp. amplitude by about a half (Onodera & Yamakawa, 1966) an effect which is also seen with iontophoretically applied pulses of acetylcholine and hence almost certainly results from a post-synaptic action of Li+ ions. While the action of Li on the endplate membrane was not the primary concern of this study, it introduced an element of uncertainty into the correction for non-linear summation of quantal voltage amplitudes (Martin, 1955). Preliminary experiments using a point clamp technique (Takeuchi & Takeuchi, 1960) revealed that the reversal potential of the transmitter immediately after changing to Li-Ringer was not very different from the value in Na-Ringer. It seems likely that the reduction in the m.e.p.p. amplitude in Li solutions is mainly due to a reduction in the conductance of the end-plate membrane during the action of the transmitter. To avoid uncertainties in estimating the mean quantal content (in) of the end-plate potential the mean end-plate potential (e.p.p.) amplitude was reduced by decreasing the [Ca2+]0 and raising the [Mg2+]. so that even after a period of growth in Li-Ringer the mean e.p.p. amplitude rarely exceeded 10 mV. Since, over the time course of these experiments Li-Ringer did not affect the resting potential of the muscle fibre non-linear summation under these conditions probably did not seriously distort the estimates of m. Unless specifically stated in the text no correction for nonlinear summation has been made for end-plates in Li-Ringer. The mean quantal content of the e.p.p. was calculated directly (del Castillo & Katz, 1954) from U

where v3 is the mean e.p.p. amplitude and a is the mean amplitude of the m.e.p.p. Estimates of the m.e.p.p. frequency (f ) entailed averaging over at least thirty events and for higher frequencies often many more than this.

RESULTS

General features of transmitter release in Li-Ringer Synaptic transmission in Li-Ringer is characterized by slow increases in both the mean quantal content ofthe e.p.p. and ofthe m.e.p.p. frequency. Immediately after changing from Na- to Li-Ringer the only effect is a reversible halving of the m.e.p.p. mean amplitude (ii). In thirty-seven experiments (il) in Li-Ringer was 0-532 + 0-127 (s.E.) of its value in NaRinger. There is no immediate effect on either the m.e.p.p. frequency or the mean quantal content. During the first hour in Li solutions both of

113 TRANSMITTER RELEASE AND Li+ these quantities slowly increase with quite reproducible and distinct time courses. Fig. 1 shows sample records of the e.p.p. at various times during an exposure to Li-Ringer and also after recovery in sodium Ringer. The results from a different experiment are illustrated in Figs. 2 and 3. Divalent cation concentrations were 0-4 mM-Oa and 3-2 mM-Mg in order to reduce the mean quantal content of the e.p.p. to about 1 at the beginning of the Li exposure. The m.e.p.p. frequency increases exponentially with time (see also Fig. 5) until spontaneous release rates are too high to be measured accurately with intracellular recording. During the first 40 min in 114-5 mM-Li-Ringer the m.e.p.p. frequency at any time (ft) is given by (1) ft = fo exp (t/r1), where fo is the m.e.p.p. frequency at the beginning of the exposure, and Tf is a constant. Tf was 10-6 + 1-1 (S.E.) minutes in 114-5 mM-Li, 0-4 mM-Ca,

B

A

A_

Il I

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C

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D n>

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wo-

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Fig. 1. Growth of the end-plate potential in Li-Ringer (1 14-5 mM). A, just after changing to Li-Ringer. B, 14 min in Li-Ringer. C, 23 min in Li-Ringer. D, 37 min in Li-Ringer. E, 43 min after returning the preparation to Na-Ringer. [Ca2+]0 0-5 mm and [Mg2+]. 4 mm throughout. Scales: horizontal 15 msec, vertical A, 1-5 mY; B, 3 mY; C, 6 mV; D, 15 mV; E, 1-5 mV. Note the changes in the vertical scale.

and 3-2 mM-Mg Ringer. The results of seven experiments are given in Table 3. After about an hour the m.e.p.p. frequency falls again producing a peak at about 50-60 min. The decline is again an exponential with a time constant of about 40 min. In the first 50 min both the mean quantal content and the m.e.p.p. frequency increase by about two orders of magnitude. Just before the peak of the m.e.p.p. frequency transmission fails at a stage when the mean quantal content is very much increased. The increase in the mean quantal content of the e.p.p. is not a simple exponential as can be seen (open circles) in the two experiments illustrated

A. C. CRAWFORD in Fig. 4. The growth- of the mean quantal content is in fact a multiplication of the extra quanta released at any time. If one subtracts the value of the mean quantal content at the beginning of the exposure (in0) from the value at any time (int) and replots the data on semilogarithmic co-ordinates (filled circles in Fig. 4) the resulting curve is a straight line described by Mt= MO +A. exp (tItm), (2) where rm is a constant and t is the time after the beginning of the Li exposure. The value of the constant A was always very small (less than 0.01) and thus a reasonable approximation to the eqn. (2) above is (3) Mt= MO+ A (exp (tI7,m) -1). Table 2 gives values of Trm and Tf in four experiments in which both evoked 114

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40 60 Minutes Fig. 2. The early time course of increases in the m.e.p.p. frequency (f ), and the mean quantal content of the e.p.p. (in) during a lithium exposure. The preparation was changed from 112-5 mM-Na-Ringer to 112-5 m s.LiRinger at the arrow. [Ca2+]0, was 0-4 mm and the [Mg2+]. was 3-2 mis throughout. Ordinate: in andf (sec-1). Abscissa: time (min). Temperature 200 C. Transmission failed in this experiment 4 min after the last point on the graph for m. 20

TRANSMITTER RELEASE AND Li+ 115 and spontaneous release was followed continuously in 114-5 mM-Li 0-4 mm-Ca, 3-2 mM-Mg-Ringer. The mean value of Tm was 8-39 + 1X22 (s.E.) min and the ratio Tm/Tt was not significantly different from 1-0. Figs. 4 and 5 illustrate one further point. It was a general finding that just before transmission was blocked the m.e.p.p. frequency began to increase more quickly than it had done during the preceding 30 min and at the same time the mean quantal content started to increase less rapidly. Actual values of the m.e.p.p. frequency at peak cannot be determined by intracellular recording but are always more than 200/sec. The area under curves, such as Fig. 4B, gives the total number of Li-releasable Li

Na 300-

A

20

20

100

0

L

0

100

200

300

Minutes

LI-Ringer

Na Ringer

100 10

0

80

160 Minutes

240

300

Fig. 3. The time course of the m.e.p.p. frequency at an end-plate exposed for over 4 hr to Li-Ringer. [Ca2+], 0-4 mm and [Mg2+]o 3-2 mm throughout. The vertical line indicates the start of the Li exposure. The data from the same experiment are plotted on linear co-ordinates in A, and on semi-logarithmic co-ordinates in B. Note the exponential rise and fall of the m.e.p.p. frequency (see-'). Lines in both graphs are fitted by eye, Tempc-rature 210 C.

A. C. CRAWFORD

116 100 r- A

10

10 1-

I

m

m

I

0-1 _

0.1 L I 0

0.01

I I I 20 40 60 Minutes Fig. 4. Two experiments (A and B) that show the time course of the mean quantal content of the e.p.p. (m) after changing to Li-Ringer. The Li exposure began at zero time. [Ca]o 0-4 mM and [Mg]. 3-2 mm throughout. Plotted on semi-logarithmic co-ordinates are: open circles the value of m at different times; filled circles, the mean quantal content after subtraction of the value in Na-Ringer, i.e. these points represent the extra quantal content of the e.p.p. seen in Li. All lines are fitted by eye. Temparature was 190C in A, and 220C in B. I

I

10

Na

30

20 Minutes

Li

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300 F

200 U

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100

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200

100

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Minutes

Fig. 5. Reversibility of the effects of Li-Ringer on the m.e.p.p. frequency. The end-plate was exposed to two periods of 114-5 m -Li-Ringer and recovered in 114-5 mM-Na-Ringer. [Ca2+]0 0-4 mn and [Mg2+]o 3-2 mM throughout. Ordinate: m.e.p.p. frequency (f) sec'. Abscissa: time (min). The junction released quanta in response to nerve stimulation throughout the experiment. Temperature 18° C.

117 TRANSMITTER RELEASE AND Li+ quanta in an end-plate. For the experiment of Fig. 4 B this is approximately 850,000 and in five experiments averaged 7-8 + 2-5 (S.E.) X 105. This value is similar to the total number of quanta that can be released in the spontaneous release mode by a variety of other treatments such as exposure to Black Widow Spider venom (Clark, Hurlbut & Mauro, 1972), La3+ ions (Heuser & Miledi 1971; Kajimoto & Pirpekar, 1972) or prolonged treatment with high Ca Ringer (Heuser, Katz & Miledi, 1971). TABLE 2. Time constants of the rise in the m.e.p.p. frequency (Tf), mins, and of the mean quantal content (Tm), min, in 0-4 mM-Ca, 3-2 mM-Mg, 114-5 mM-Li-Ringer

Expt. 1 2 3 4 Mean + s.E.

Tm (min)

8-25 10-26 5-00 10-05 8-39 + 1-22

Tf (min)

5-5 12-7

8-9 11-3 9-60 + 1-56

Tm/Tf

1-50 0-81 0-56 0-89 0-94 + 0-20

Provided that the m.e.p.p. frequency has not gone through its peak, changes in spontaneous and evoked release transmitter produced by LiRinger are completely reversible. This is illustrated in Fig. 5 which shows an experiment in which an end-plate was exposed to two periods of LiRinger and recovered in between in Na-Ringer. Both the mean quantal content of the e.p.p. and the m.e.p.p. frequency return to their initial values in about 20 min after the first Li pulse and rise with the same time constant again during the second Li exposure. Recovery in Na-Ringer is always more rapid than the increases in transmitter release that occur during the preceding period in Li. If an end-plate is exposed to Li-Ringer for periods long enough to cause transmission block it is not possible to recover the e.p.p. and the m.e.p.p. frequency declines only very slowly. It appears that after about 40 min in Li-Ringer irreversible changes take place. The failure of synaptic transmission A question of immediate interest is whether the failure of synaptic transmission is due to interference with the presynaptic action potential or whether it results from a failure of the release mechanism itself. This problem can be tackled directly by recording the nerve action potential and synaptic currents extracellularly at a single nerve terminal. Fig. 6 shows sample records of focally recorded action currents and end-plate currents (e.p.c.s.) at a single active spot within a junction. All such measurements were made with electrodes filled with 0-5 M-Li Cl instead of the more usual NaCl (Katz & Miledi, 1965), to avoid introducing Na+

A. C. CRAWFORD ions into the immediate vicinity of the terminal and thereby distorting the time course of the Li effects. The position of the focal recording electrode is of course crucial; the space constant of the extracellular currents is very small and small movements of the electrode away from an active spot often lead to loss of the nerve action potential and a reduction in the amplitude of the e.p.c. However, in five experiments it was possible to record nerve action currents and e.p.c.s. up to transmission block. In all cases both 118

A

B

L

Fig. 6. Failure of synaptic transmission in Li-Ringer. A, focal records of nerve action potentials and end-plate currents after 39 min in Li-Ringer. [Ca2+]0 0-4 m , [Mg2+]. 3-2 mM. B, records from the same spot 3 min later. Note the loss of nerve action potentials and end-plate currents together. The lowest trace in B shows a spontaneous miniature end-plate current. Scale: 1 msec and 0-5 mV. Temperature 21° C.

nerve action current and e.p.c. failed together. Movement of the electrode away from the terminal could spuriously produce this result but it seems unlikely for two reasons. Loss of the nerve action current and e.p.c. occurred at about the same time in all five experiments (37 + 6 (s.E.) min) and at a time when transmission block was found to occur in experiments

TRANSMITTER RELEASE AND Li+ 119 with intracellular recording. Furthermore, the amplitude of the miniature e.p.c.s. immediately before and after loss of the nerve action current and e.p.c. was not significantly changed. Movement of the electrode away from the active spot would tend to reduce the mean miniature e.p.c. amplitude. It seems probable then that transmission failure results from the failure of the nerve action potential to invade the motor nerve terminals and does not necessarily involve a defect in the release mechanism itself. This conclusion is supported by the behaviour of the latency of evoked transmitter release before failure occurs. 10

1

8

E

4 2 0

0

10

20 Time (min)

30

40

Fig. 7. Changes in synaptic delays during an exposure to Li-Ringer. Lower curve: true synaptic delay (from the peak of the action potential to the onset of the end-plate current). Upper curve: total response latency. The time difference between the two curves is the action potential conduction delay. The preparation was exposed to 114-5 Li+, 04 Ca2+, 3-2 Mg2+ Ringer at zero time. Ordinate: synaptic delays (mnsec). Abscissa: time (min). The arrow indicates the time at which synaptic transmission failed. Temperature 190 C.

It was noticed with intracellular recording that the total response latency, i.e. the time between stimulation and the onset of the e.p.p., increased considerably before conduction block occurred. The phenomenon was pursued in detail, however, only in the experiments using focal recording where it was possible to separate the conduction delay (time to the invasion of the terminal by an action potential) from the true synaptic delay (the lag between the arrival of the nerve action potential and the onset of the e.p.c.). Fig. 7 illustrates how these two delays change with time after the beginning of a Li exposure. The arrow indicates the

A. C. CRAWFORD 120 time at which both e.p.c. and nerve action current were lost. While at room temperature the changes in the synaptic delay (about 0 5 msec) cannot be measured very accurately, it is clear that most of the increase in the total response latency results from an increase in the conduction delay. This increase in the time taken for an action potential to invade the terminal, presumably due to a reduced conduction velocity, is most marked immediately before transmission block. In view of the fact that it is known that Li+ ions are extruded from cells only very slowly by the Na pump (Flynn & Maizels, 1950; Keynes & Swan, 1959), and that significant accumulation may therefore occur (Carmeliet, 1964; Niedergerke & Orkand, 1966), it seems reasonable to explain the decrease in the conduction velocity of the action potential and the ensuing conduction block by such an accumulation of Li+ ions inside the nerve.

The extent of the m.e.p.p. frequency at peak In the presence of a cholinesterase inhibitor such as prostigmine the m.e.p.p. has such a long duration that events occurring at a rate of more than 200/sec cannot be reliably distinguished. If one records the miniature end-plate currents from an active spot it is possible to follow much higher spontaneous release rates not only because the duration of the event is much shorter but also because the focal electrode records from only a small fraction of the area of the end-plate (Fatt & Katz, 1952). The m.e.p.c. frequency throughout two experiments in which end-plates were exposed to Li-Ringer is illustrated in Fig. 8. Peak m.e.p.c. frequencies were rather variable (range 5-14/sec), probably the result of recording from different areas of the end-plate. Heuser & Miledi (1972) estimate that a focal electrode records from 1/20 to 1/50 of the total end-plate area. In three experiments an intracellular electrode was inserted into the end-plate after the peak m.e.p.c. frequency had been reached and the ratio of the intracellularly recorded m.e.p.p. frequency to the focally recorded m.e.p.c. frequency was determined. This allowed the peak m.e.p.p. frequency to be calculated. The values in the three experiments were 376, 420 and 463/sec. An accurate knowledge of the peak m.e.p.p. frequency turns out not to be crucial for the determination of the total number of Li-releasable quanta since the peak occupies a very short time. The Ca dependence of change in spontaneous release In view of the eventual conduction block in Li-Ringer it seems likely that depolarization of the nerve terminals may contribute to changes in the m.e.p.p. frequency. Ringer solutions that contain no added Ca together with a Ca chelating agent such as EGTA are known to abolish depolariza-

121 TRANSMITTER RELEASE AND Li+ tion-induced increases in the m.e.p.p. frequency (Hubbard, Jones & Landau, 1968a; Cooke, Okamoto & Quastel, 1973). Nine experiments were carried out in which the Ringer contained 1 mM-EGTA, 5 mM-Mg and no added Ca. The major cation was either 112 mM-Na+ or 112 mM-Li+. 10 6

B

A

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v80 40 120 0 40 80 120 Minutes Minutes Fig. 8. Time course of the frequency of focally recorded miniature end-plate currents (f, sec-1) during a long exposure to Li-Ringer. Two experiments (A and C) are illustrated. Graphs B and D show the same data on semilogarithmic co-ordinates. The preparations were changed to Li-Ringer at zero time. [Ca2+]0 0-4 mx and [Mg2+]o 3-2 mx throughout. Ordinates: m.e.p.c. frequency. Abscissa: time (min). Note the exponential rise and fall of the m.e.p.c. frequency. Temperatures 19° C (A and B) and 220 C (C and D). 0

Fig. 9 shows the time course of the m.e.p.p. frequency during a long exposure to Li-Ringer and in the absence of Ca ions. The m.e.p.p. frequency rises initially with a very similar time course to that in experiments where Ca is present. A plateau is then reached at a frequency of about 60/sec

A. C. CRAWFORD 122 from which spontaneous release slowly declines over the next few hours. The massive spontaneous release rates seen during the m.e.p.p. frequency peak in the presence of Ca are never observed. The initial rise in the m.e.p.p. frequency follows an exponential time course not significantly different from that in the presence of external Ca. Fig. 10 shows on semilogarithmic co-ordinates the initial time courses of A, eight experiments in 0 4 mM-Ca (Li) Ringer and B, seven experiments in O-Ca, 1 mM-EGTA (Li) Ringer. It can be seen that for the first 30 min of a Li exposure the Na (EGTA)

Li (EGTA)

60

U

f40

20

10 80 120 Time (min) Fig. 9. Time course of the m.e.p.p. frequency (f) sec-1 in Li-Ringer in the absence of external Ca ions. The preparation was changed to Li-Ringer from Na-Ringer at the vertical line in the Figure. Both solutions contained no added Ca and 1 P.mmEGTA. [Mg2+],, was 3-2 mm throughout. Note the exponential rise of the m.e.p.p. frequency to a maximum of about 60 sec-1 and the subsequent slow decline. Temperature 19' C. 0

'40

m.e.p.p. frequency is independent of external Ca. Table 3 gives values for Tf in seven experiments in 0-4 mm-Ca Ringer and seven different experiments in EGTA Ringer. The [Li]0 was 114-5 mm throughout. The mean value of Tf in the presence of Ca ions (10- 64 ± 1 lIO (s. E;.) min) was not significantly different from that in the presence of no added Ca and of 1 mm-EGTA (I11 40±+I17 6 (s. E.) min). After the initial 30 min period, however, a different picture is seen. Late in Li exposure spontaneous release is greatly accelerated by a process dependent on external Ca. Fig. 11I illustrates an experiment in which an end-plate was exposed to a pulse of Ca-free (EGTA) Ringer late in an exposure to Li (0-4 mm-Ca) Ringer. The m.e.p.p. frequency is stabilized by removing external Ca and experiences an immediate and rapid

TRANSMITTER RELEASE AND Li+ 123 rise when Ca is again added to the bathing solution at the end of the EGTA pulse. Fig. 12 shows the complementary experiment where an end-plate was first exposed to Li in EGTA Ringer and a pulse of Ca applied as indicated between the vertical lines. The m.e.p.p. frequency is greatly elevated by the presence of external Ca and on returning to EGTA Ringer it continues to increase. A Na-Ringer

[I

B Na-Ringer

Li-Ringer

Li-Ringer

!I

100

10 0-1

U

1 I0

0

0 10 20 10 20 30 40 30 Time (min) Time (min) Fig. 10. The rise in the m.e.p.p. frequency in Li-Ringer is exponential and independent of the external calcium concentration. A and B are semilogarithmic plots of the m.e.p.p. frequency (f, ordinates) sec- against time (abscissa, min) for experiments where the preparation was changed from 114-5 mk-Na-Ringer to 114-5 m -Li-Ringer at the vertical line in each diagram. Experiments of A were carried out in 04 m -Ca, 3X2 mM-Mg throughout. In the experiments of B the Ringer contained no added Ca and 1 mM-EGTA. [Mg2+]. was 3-2 m . All experiments at room temperature (18-220 C).

TABLE 3. Time constants of the rise in the m.e.p.p. frequency in 114-5 mM-Li-Ringer (min). [Mg2+]. 3-2 mm throughout

Expt. 1 2 3 4 5 6 7 Mean + s.E.

0-4 mM-Ca

1 mim-EGTA

5-5

11-0 14-7 20-0 6-8 9.5 10-8 7*0 11*40 + 1-76

9-5 10-5

12*7 10*8 10-5 15-0 10*64+ 1.10

124 A. C. CRAWFORD It seems reasonable to attribute this sensitivity of spontaneous release to the external Ca2+ ions late in the Li exposure to depolarization of the nerve terminals. Its onset (after 30 min) occurs at about the time when the conduction velocity of the nerve action potential is increasing and is most dramatic after conduction block has occurred. Li

(0.4 Ca) Na (0.4 Ca) 150

Li (0.4 Ca)

Li (EGTA)

Na (0.4 Ca)

100

o

L 0

20

40

60 80 100 120 140 Time (min) Fig. I 1. The effects of removing external Ca late in a Li exposure. Ordinate: m.e.p.p. frequency (f) sec-1. Abscissa: time (min). The preparation was exposed to Li-Ringer at the first vertical line in the Figure. The whole experiment was run in 0-4 mM-Ca, 3-2 mM-Mg, except for a short period (labelled Li (EGTA) in the diagram) when external Ca2+ ions were removed by flowing Li-Ringer with no added Ca and 1 mM-EGTA (3-2 mM-Mg) over the preparation. Note that the 'Ca-free' pulse reduces and slows the m.e.p.p. frequency rise but on returning to Li (Ca) Ringer spontaneous release dramatically accelerates. Temperature 200 C.

One interesting point in these experiments is that the m.e.p.p. frequency shows no evidence of recovery when Ca is withdrawn from the Ringer. Changing the [Ca]. around K-depolarized terminals produces only reversible changes in the m.e.p.p. frequency (Hubbard, Jones & Landau, 1968 a; Cooke, Okamoto & Quastel, 1973). Terminals late in a Li exposure are Ca-sensitive but irreversibly so.

Tetanization of the presynaptic nerve in Li-Ringer Changes in spontaneous and evoked transmitter release induced by Li are extremely slow, quite unlike the immediate changes induced by replacing part of the Na of the Ringer by non-polar substitutes (Kelly, 1965, 1968; Colombo & Rahamimoff, 1968). Quite possibly, Li accumulation inside the nerve terminals may underlie changes in transmitter

TRANSMITTER RELEASE AND Li+ 125 release. Applying a tetanus to the nerve in Li-Ringer ought to accelerate Li accumulation and provide evidence for such an effect. Nerve terminals were bathed in 1 mm-EGTA Ringer to avoid large changes in the m.e.p.p. frequency due to Ca entry during tetanization (Katz & Miledi, 1968; Rosenthal, 1969; Weinreich, 1971; Miledi & Thies, 1971). An experiment in which 3000 stimuli at 50 Hz were presented to the nerve is illustrated in Fig. 13. The preparation was exposed to LiRinger at zero time on the graph. The tetanus produces a massive increase Li (EGTA)

Li (0.4 mM- Ca)

Li (EGTA)

100

UI

4-

10

I 0

10

20

30 Time (min)

40

50

Fig. 12. Ca sensitivity of the m.e.p.p. frequency late in a Li exposure. Ordinate: m.e.p.p. frequency (f) sec'. Abscissa: time (min). Note the semilogarithmic co-ordinates. The preparation was changed to Li (EGTA) Ringer at zero time in the Figure. Between the vertical lines in the diagram the end-plate was exposed to a Ringer containing 04 mM-Ca. [Mg2+]. 3-2 mM throughout. Note the sudden elevation of f in Li(Ca) Ringer. The time constant of the rise of the m.e.p.p. frequency is not significantly different after the Ca2+ exposure from the value before. Temperature 220 C.

in the m.e.p.p. frequency; in Na-Ringer such procedures produce about a doubling of the rate of spontaneous release (Miledi & Thies, 1971). Just how sensitive the preparation has become to stimulation can be seen from Fig. 14B. In this experiment only 100 and 200 pulses were applied at T1 and T2 respectively, both at a frequency of 5 Hz. The tetani both produce two effects: (a) a steep increase in the m.e.p.p. frequency that

126 A. C. CRAWFORD occurs during the tetanus and (b) an increase in the time constant of the subsequent rise in the spontaneous release rate. A further surprising point is that there is no evidence of any recovery of the m.e.p.p. frequency at the end of the tetanus: the m.e.p.p. frequency can be raised to a higher rate by stimulating the nerve but recovery to the original value, as occurs after tetani in Na-Ringer, is never seen.

100

10

0

8

16

24 32 40 Time (min) Fig. 13. The effect of tetanizing a preparation on the m.e.p.p. frequency in Li (EGTA) Ringer. Ordinate: m.e.p.p. frequency (f) sec'. Abscissa: time (min). Note the semilogarithmic co-ordinates. At the arrow and bar a tetanus of 3000 stimuli at 50 Hz was applied to the nerve. The Ringer contained no added Ca, 1 mM-EGTA, and 3-2 mM-Mg throughout. Note the exponential rise in the m.e.p.p. frequency before the tetanus and the hundredfold rise during the tetanus. Temperature 200 C.

One question that arises is whether the Li entry during the tetanus is important at all; a tetanus might merely test the state in which the resting entry of Li before the tetanus has left the end-plate. This does not seem to be the case. Identical tetani produce the same multiplication of the m.e.p.p. frequency independent of the stage of the Li exposure at which they occur and independent of the presence of preceding tetani. Fig. 14A illustrates an experiment in which a tetanus of 600 pulses at 50 Hz was given at two different times during the exposure of an end-plate to Li (EGTA) Ringer. The step in the m.e.p.p. frequency that occurs during the tetanus is the same multiplication for both tetani. What characteristics of the tetanus are important in producing the

127 TRANSMITTER RELEASE AND Li+ increases in spontaneous release? In Na-Ringer in the presence of Ca, changes in the m.e.p.p. frequency are very sensitive to the frequency of the tetanus; a single shock accelerates the m.e.p.p. frequency only transiently (Rahamimoff & Yaari, 1973) which presumably accounts for the fact that Hubbard (1963) found frequencies of stimulation lower than 10 Hz ineffective in .changing the rate of spontaneous release. In Li (EGTA) Ringer the situation is rather different. In the experiment of Fig. 15 an end-plate was given two tetani, one at 5 Hz and the other at 50 Hz, both 500 pulses in duration, during a period in Li (EGTA) Ringer. There is little difference between the responses of the m.e.p.p. frequency during the two tetani. In three similar experiments a 600 pulse tetanus at 5 Hz increased the m.e.p.p. frequency by 1-43 + 0-12 (s.E.) of its initial value while a 50 Hz tetanus for 600 pulses increased the B

A

100

100 U

'u

10

W

10

a,

T2

T2

I~~t

I

L0*1

0.1

I

I

I

40 0 10 30 20 20 30 40 Minutes Minutes Fig. 14. A, identical tetani produce the same multiplication of the m.e.p.p. frequency no matter at what time in a Li exposure they are applied. Ordinate: m.e.p.p. frequency (f) sec-'. Abscissa: time (min). The preparation was in a Ringer that contained no added Ca, 1 mM-EGTA, and 3*2 mM-Mg throughout. The nerve was stimulated at a rate of 50 Hz for 600 pulses at the arrows labelled T, and T2 in the Figure. Note the same multiplication of the m.e.p.p. frequency occurs during each tetanus. B, even short tetani in Li (EGTA) Ringer produce significant acceleration of the m.e.p.p. frequency. Ordinate: m.e.p.p. frequency (f ) sec-". Abscissa: time (min). Note the semilogarithmic co-ordinates. The preparation was stimulated at a rate of 5 Hz three times as indicated by the arrows at T1, T2 and T3. T1 consisted of 100 pulses, T2 and T3 of 200 pulses. Note that the multiplication of the m.e.p.p. frequency is twice that of T1 in response to T2. T3 did not produce any effect probably because nerve conduction had been blocked by this time. Ringer contained no added calcium 1 mM-EGTA, and 3-2 mM-Mg. Temperatures: A, 220 C; B, 19° C.

0

5

10

PHY 246

128 A. C. CRAWFORD m.e.p.p. frequency by 1960 + 0-17 (S.E.) of its value at the beginning of the tetanus. What is important in determining the increase in the m.e.p.p. frequency is the number of pulses in the tetanus. The two tetani of Fig. 14B were both at a frequency of 5 Hz but T1 is 100 pulses and T2 200 pulses long. Clearly the multiplication of the m.e.p.p. frequency is larger in T2 than in T1. In three experiments of this type 200 pulses (5 Hz) produced a multiplication of the initial frequency of 1921 + 007 (S.E.) while a 600 pulse (5 Hz) tetanus gave a multiplication of 1-43+ 0-07 (S.E.). T.

T2

100

10

--

7

0*1~~~~~~~~~~~~~~0

0

20

40 60 Minutes Fig. 15. Multiplication of the m.e.p.p. frequency in Li (EGTA) Ringer by a tetanus is insensitive to the frequency of stimulation. Ordinate: m.e.p.p. frequency (f) sec-1. Abscissa: time (min). Two tetani (T1 and T2 in the Figure) were applied to the preparation as indicated by the vertical lines. T1 consisted of 500 pulses at a rate of 5 Hz and T2 of 500 pulses at a rate of 50 Hz. Note that the same multiplication of the m.e.p.p. frequency occurs in both tetani. Preparation in a Ringer that contained no added Ca, 1 mM-EGTA and 3*2 mM-Mg, throughout. Note the semilogarithmic co-ordinates. Temperature 180 C.

These observations suggest an accumulation effect, each stimulus adding an accelerating increment to the m.e.p.p. frequency without significant decay of the activating effect between pulses. Such a process would also explain why the m.e.p.p. frequency does not recover at the end of a tetanus. It seems likely that accumulation of Li ions is directly or indirectly responsible for this process.

TRANSMITTER RELEASE AND Li+

129

Li-Na mixtures and the time course of changes in transmitter release If the slow time course of the increases in transmitter release seen in 114-5 mm-Li Ringer is due to Li accumulation in the terminals, it is of interest to examine the effects of lower concentrations of external Li on the properties of release. If only half of the Na of the Ringer is replaced by Li as in Fig. 16 the m.e.p.p. frequency continues to increase exponentially, but the time constant is more than twice that seen in 100 % LiRinger. If left long enough in 50 % Li-Ringer the m.e.p.p. frequency increases to the same extent as it does in 100 % Li-Ringer but does so more slowly. The [Li+]o affects the time course of the increases in the 20

10

-

,

A

T~~~ 0

5

2 !

0

I

I

20

I

I

40

I

l

I

60

Time (min) Fig. 16. Time course of the rise in the m.e.p.p. frequency (four experiments) in 50 % Li 50 % Na Ringer. Ordinate: m.e.p.p. frequency (f ) sec-1. Abscissa: time (min). Note the semilogarithmic co-ordinates. The preparations were changed from Na-Ringer to 50 % Li-Ringer at zero time on the graph. [Ca2+]o 0-4 m and [Mg2+1] 3*2 mm throughout. Temperature 210 C.

m.e.p.p. frequency but the [Ca2+]o does not. Time constants for experiments in 50 % Li-Ringer are also included in Table 4. The data for Tom, the time constant of the increase in the extra mean quantal content of the e.p.p., are also included. It does not appear that in Ringer containing 50 % Li and 50 % Na, the [Li+]o limits the extent of changes in transmitter release. The results would suggest that only small amounts of Li need be accumulated inside the terminals to produce considerable increases in transmitter release. This view is supported by the observation that as little as 100 stimuli can 5-2

130 A. C. CRAWFORD produce a significant increase in the m.e.p.p. frequency in Li (EGTA) Ringer. The time course of changes in transmitter release at [Li+]o less than 56 mm was too slow to follow properly in unstimulated preparations. It was, however, possible to examine the effects of Ringer that contained only a few millimolar Li by stimulating the nerve to accelerate Li entry. Fig. 17 illustrates an experiment in which only 10 % of the Na of the Ringer had been replaced by Li. A control tetanus at 18 Hz was performed in Na-Ringer before the exposure to 10 % Li-Ringer and this produced only a small increase in the m.e.p.p. frequency and mean amplitude of the TABLE 4. Time constants in 50 % Li 50 % Na Ringer. [Ca2+]. 0 4 mm and [Mg2+]. 3*2 mm throughout. All time constants in mm

Expt. 1 2 3 4 Mean+s.E.

rm (mn) 32-0

Tr (min) 25.3

31-2

28-5

26-3 43.9 33-35+3-74

28-0 57-0 34-7 + 7.46

TmITf 0-79 0.91 1-06 1-30

1-01+0-11

80

T4

T2T

60 40

1

sL1 ~L.LLL.....L... 0 10 20 30 130140

150

160

170

180

Time (min)

Fig. 17. 10% Li (Na) Ringer produces a large increase in post-tetanic potentiation of both the m.e.p.p. frequency and of the mean amplitude of the e.p.p. Ordinates: m.e.p.p. frequency (f) sec-1, and mean amplitude of the e.p.p. (v) mV. Abscissa: time (min). Filled circles indicate v, and open circles indicate f. The preparation was changed from Na-Ringer to a Ringer in which 10 % of the Na+ had been replaced isotonically by Li+ at the dotted vertical line. Three tetani were applied to the nerve all at a rate of 18 Hz. T4 is a 3 min control tetanus in Na-Ringer. T2 and T3 are test tetani applied in 10 % Li-Ringer and lasted 3 and 5 min respectively. Note the contracted time scale between 30 and 130 min. An exposure of about 2 hr to 10 % Li-Ringer produces little change in the basal level of release but the responses to tetani are modified.

TRANSMITTER RELEASE AND Li+ 131 e.p.p. The subsequent post-tetanic potentiation of both modes of transmitter release decays over several minutes (Hubbard, 1963; Rosenthal, 1969; Magleby, 1973). On changing to 10 % Li-Ringer there was no detectable change in the mean quantal content of the e.p.p. or of the m.e.p.p. frequency which occurs during the following 2 hr. Note the contracted time scale in Fig. 17 over this period. When the control tetanus (18 Hz for 3 min) was again applied (T2 in Fig. 17) rather different responses occurred. Transmitter release was not followed during the tetanus, but at the end of the tetanus a very large post-tetanic potentiation (PTP) of both the mean e.p.p. amplitude and the m.e.p.p. frequency is seen. A second tetanus (T3) at 18 Hz for 5 min produced an even larger PTP of the mean quantal content and the m.e.p.p. frequency. In four experiments the time constant of the decay of the PTP in 10 % Li-Ringer was 2-52 + 0-51 (s.E.) min for the m.e.p.p. frequency and 1'70 + 0-34 (s.E.) for the decay of the mean quantal content of the e.p.p. There was always a slight elevation of the resting release rate over the control value after PTP had completely decayed. This effect is not very large. In the experiment of Fig. 17, for instance, the mean quantal content of the e.p.p. was raised to 1-55 times its value in Na-Ringer at the beginning of the experiment, and the m.e.p.p. frequency to 1-8 times the control value. The inclusion of small amounts of Li in the Ringer has little effect on resting levels of release but significantly alters the response of an end-plate to a long tetanus. Defining the potentiation of the mean quantal content (Pm) and the potentiation of the m.e.p.p. frequency (Pf) in a similar way to Magleby (1973), p

mmax -MO mO

and

p _ fmax-fo

where mmax and fmax are the values of the mean quantal content and the m.e.p.p. frequency respectively at the end of the tetanus, and mO and fo are the corresponding values at the beginning of the tetanus. Mean values of Pm and Pr are given in Table 5 for four experiments with the same protocol as that of Fig. 17. It was not possible to detect changes in PTP when the [Li+]o was reduced to 2-5 mm. In contrast to the rather dramatic effects of even small amounts of Li on PTP, facilitation is only slightly affected by the presence of even large concentrations of Li+ ions. An experiment in which facilitation was examined by giving a double shock to the nerve at 15 sec intervals throughout the first 30 min in Li-Ringer is illustrated in Fig. 18. The double stimulus consisted of two pulses separated by

A. C. CRAWFORD

132

25 msec. Standard error of facilitation was calculated according to Rahamimoff (1968). The Ringer contained 0-4 mM-Ca and 3-2 mM-Mg. Both ml and m2 increase during the lithium exposure and facilitation which at the beginning of the experiment was too small to be distinguished (m2/mL = 1) grows slightly at first and then appears to assume a constant value when both ml and m2 are large. This experiment (Fig. 18) gave the largest increase in facilitation of three experiments in Li-Ringer. In one of the others there was little facilitation at all. If Li-Ringer does produce an increase in facilitation it is small and possibly masked by the fact that facilitation is known to decrease as the mean quantal content of the first e.p.p. (inm) increases (Rahamimoff, 1968; Mallart & Martin, 1968). TABLE 5. Potentiation of the mean quantal content (Pm) and of the m.e.p.p. frequency (Pi) in response to tetani of 18 Hz in Na-Ringer and Na (10 % Li) Ringer. See text for definitions of Pm and Pf

Na (10%) Li Ringer A Na Ringer , 3 min, 18 Hz 3 min, 18 Hz 5 min, 18 Hz 2-42+0-34 0-38+0-13 2-04+0-21

Duration P1 (±S.E.) n =4

Pm (±S.E.)

0-46 + 020

2-89 + 0-57

3-05 + 062

n =4

20

A -0-0-

3

15

m

1)

1

4.

-0-~~~~0

0

0

30 10 20 Minutes Fig. 18. Facilitation in Li-Ringer. Two stimuli 25 msec apart were presented to the nerve at 15 see intervals during a period in 114-5 mM-Li, 0-4 mM-Ca, 3-2 mm-Mg Ringer. The graph labelled A shows the time course of the growth of the mean quantal content (in) of the responses to the first (filled circles) and the second (open circles) stimuli. In B the time course of facilitation (i.e. the ratio of the second quantal content to the first, F) is indicated. The preparation was changed from Na-Ringer to Li-Ringer at zero time in both diagrams. The vertical bars in B are + 1 S.E. of F as defined by Rahamimoff (1968). Horizontal bars in both A and B indicated the time period over which e.p.p.s were recorded to give the trials for a single value of a mean quantal content. Temperature 200 C. 0

10 20 Minutes

30

0

TRANSMITTER RELEASE AND Li+

133

Depolarization and the action of Li One of the most puzzling aspects of the increases in transmitter release that occur in Li-Ringer is their exponential time course. If Li enters the terminals by moving down its electrochemical gradient and the permeability of the terminals to Li is increased by depolarization, then it is conceivable that a progressively accelerating m.e.p.p. frequency might result from a steadily increasing influx of Li. However, any explanation based on a voltage-sensitive Li permeability is hard to reconcile with experiments of the type illustrated in Fig. 19. In the middle of an exposure to Li-Ringer an end-plate was subjected to a K depolarization of 20 mM-K+. Raising the [K+]. by this amount (2-20 mM) was found in a number of experiments to block synaptic transmission presumably because the conduction of the action potential in the presynaptic nerve has failed (Hubbard & Willis, 1968). Such a treatment depolarizes the terminals to a greater extent than does a period of 30 min in Li-Ringer for transmission has usually not failed by this time. The [Li+]o was kept constant in these experiments at 97-5 mm and Tris used to maintain isotonicity. Elevation of the [K+]0 was achieved by isotonic replacement of Tris by K. The full composition of the solutions is given in columns D, E and F of Table 1. The whole experiment was run in EGTA (Ca-free) Ringer to avoid Cadependent changes in the m.e.p.p. frequency that occur when end-plates are depolarized (Hubbard et al. 1968a; Cooke et al. 1973). In order to maintain the m.e.p.p. amplitude large enough to carry out accurate measurements of the m.e.p.p. frequency in Ringer that contained a high [K+]., EGTA and were Li based, it was found necessary to replace the Cl of the Ringer by large impermeant ions (see Methods). It is clear from Fig. 19 that a K depolarization applied during a Li exposure actually reduces the m.e.p.p. frequency, which then continues to rise with a much slower time constant than in 2 mM-K (Li) Ringer. On returning to 2 mM-K (Li) Ringer the m.e.p.p. frequency accelerates again and when the Li of the Ringer is replaced by Na the end-plate partially recovers. Essentially similar results were obtained from two other experiments with the same protocol as Fig. 19. It does not seem from these experiments that the rise in the m.e.p.p. frequency or its exponential time course results from depolarization per se. If the Li permeability of the terminals were roughly constant during the first 30 min of a Li exposure then an imposed depolarization such as that of Fig. 19 ought to slow the rate of Li entry and hence presumably the rate of rise of the m.e.p.p. frequency. This does seem to happen: the time constant of the rise in the m.e.p.p. frequency in 20 mM-K (Li) Ringer is about 5 times that in 2 mM-K (Li) Ringer.

134

A. C. CRAWFORD 100

U 01

Li (2K)

Li (20K)

Li (2K)

Na (2K)

10

0

10

20

30 40 Minutes

50

60

70

Fig. 19. Depolarization of the nerve terminals does not underlie the effects of Li on spontaneous release. An end-plate was depolarized with 20 mm-K+ Ringer in the period labelled 'Li(20K)' in the Figure. All solutions contained no added Ca and 1 mM-EGTA. The [Mg2+]. was 3-2 mm throughout. At the first vertical line the preparation was changed to 114-5 mM-LiRinger and the m.e.p.p. frequency rises exponentially. The double vertical lines in the figure delimit the beginning and end of subsequent solution changes. The change to 20 mM-K (Li) Ringer was carried out especially slowly to prevent contraction of the muscle when it was depolarized. Note the reduction of the m.e.p.p. frequency in the high-K+ Ringer and the slowing of its subsequent rise. Ordinate: m.e.p.p. frequency (f) sec-1. Abscissa: time (min). Note the semilogarithmic co-ordinates. Temperature 210 C. DISCUSSION

In Li-Ringer transmitter release is characterized by slow increases in both the m.e.p.p. frequency and the mean quantal content of the end-plate potential. The increase in spontaneous release of transmitter can be divided into two phases; during the first 30 min of a Li exposure the m.e.p.p. frequency accelerates exponentially with time and independently of the [Ca2+]o while at times subsequent to this an extra component of m.e.p.p. frequency is added that is dependent on the presence of external Ca. During the early phase of spontaneous release the mean quantal content of the end-plate potential grows by the addition of extra quanta, the

TRANSMITTER RELEASE AND L1+135 increment growing exponentially with time and with a time constant identical to that of the increase in the m.e.p.p. frequency. The first phase of the increase in the m.e.p.p. frequency is unlikely to be caused by Ca entry into the terminals either by depolarization or by activation of a Na-dependent Ca influx since it occurs in Ringer containing EGTA. The acceleration of spontaneous release during the second phase is probably due to depolarization of the presynaptic terminals because (i) the conduction velocity of the action potential in the nerve is slowed, (ii) the presynaptic spike eventually fails, and (iii) the m.e.p.p. frequency increases more rapidly to a peak of several hundred quanta second provided that Ca is present in the Ringer. Perhaps the most interesting aspect of these results is the exponential increase in both evoked and spontaneous release that occurs in the first phase of a Li exposure. It seems clear that the increase in the m.e.p.p. frequency is dependent on the entry of Li into the nerve terminal because (i) the m.e.p.p. frequency can be accelerated irreversibly by a tetanus even in EGTA-(Li) Ringer, (ii) the magnitude of the increase in spontaneous release during a tetanus depends on the number of pulses in the tetanus and not on the frequency, and (iii) the time constant of the increase in the m.e.p.p. frequency (and of the increment in m) is strongly dependent on the [Li+]o - the lower the [Li+]o the slower the increase inf. It should be pointed out that entry of Li into the terminals presumably occurs in exchange for internal cations and the fall in [Na+]i and [K+]i may be the primary mediator of the effects on transmitter release. The similarity of many of the results presented here to the action of cardiac glycosides on transmitter release (Birks & Cohen, 1968a, b) raises the possibility that in both situations a falling intracellular K concentration within the presynaptic terminal may affect the release of transmitter. Certainly the observation that raising the [K+]. dramatically slows the increase in the m.e.p.p. frequency in Li-Ringer, is consistent with this. But how can changes in the intracellular concentration of Li or other monovalent cations influence the release of transmitter? There seem to be three types of explanation: (i) internal Li+ ions may act as direct activators of release similar to Ca or (ii) the monovalent cation concentrations inside the presynaptic terminals may change the effectiveness of internal Ca2+ ions in releasing quanta or (iii) internal stores of Ca could be liberated by the entry of Li into the nerve terminals. The first hypothesis cannot be entirely ruled out but it seems unlikely in view of the fact that very few ions will replace Ca in sustaining the end-plate potential. Really only Sr and to a lesser extent Ba are effective in maintaining evoked transmitter release when Ca is omitted from the external solution (Dodge, Miledi & Rahamimoff, 1969; Katz & Miledi, 1969; Meiri & Rahamimoff, 1972)

136 A. C. CRAWFORD while other divalent cations such as Mg2+, Be2+, Mn2+ and C02+ act as inhibitors of release (Dodge & Rahamimoff, 1967; Hubbard et al. 1968b; Blioch et al. 1968; Balnave & Gage, 1971; Blioch & Liberman, 1970; Kajimoto & Pirpekar, 1972; Meiri & Rahamimoff, 1972). In the experiments reported here there was never any indication that evoked release could be sustained in Li (EGTA) Ringer or that Li ions could exert an inhibitory effect on the mean quantal content of the e.p.p. when Ca was present. It is difficult to see at present how the second hypothesis above can be investigated further: but the third explanation - that Li releases Ca from stores inside the nerve terminals - is attractive because it explains many

of the findings presented here. Most of the Ca in nerve axoplasm is bound within the mitochondria (Baker, 1972). This Ca store is not inert; the distribution of Ca between axoplasm and mitochondria is maintained in a steady state where a slow leak of Ca out of the mitochondria is balanced by active uptake (Lehninger, Carafoli & Rossi, 1967; Blaustein & Hodgkin, 1969; Baker, Hodgkin & Ridgway, 1971). In squid nerve the mitochondria not only provide a large store of Ca that can be released into the xxoplasm but also play an important role in controlling the Ca concentration of the axoplasm when Ca ions enter the nerve through the surface membrane (Baker, Meves & Ridgway, 1973). An inhibitory action on the mitochondrial Ca-sequestering system as a result of Li entry into the nerve terminals might explain why the m.e.p.p. frequency is irreversibly raised by adding Ca to the Ringer late in a Li exposure (see Fig. 12), and why post-tetanic increases in both the e.p.p. amplitude and the m.e.p.p. frequency are larger if some of the Na of the Ringer is replaced by Li. End-plates in Na-Ringer that are depolarized by exposure to high K solutions show an elevation of the m.e.p.p. frequency if Ca is present but the spontaneous release rate recovers to its control value after Ca has been withdrawn (Hubbard et al. 1968a; Cooke et al. 1973). It seems probable that the m.e.p.p. frequency is raised under these conditions by entry of Ca into the presynaptic terminals (Hubbard, 1973) and the recovery is due to the sequestering or extrusion of the calcium that has entered. The failure of the m.e.p.p. frequency to recover from a Ca challenge late in a Li exposure may indicate that the Ca sequestering mechanisms are then less effective. The increases in PTP in Li-Ringer may have a similar basis for Weinreich (1971) and Rosenthal (1969) both observed that the [Ca2+]o during the tetanus was most important for the development of PTP while Rosenthal (1969) found that the decay of PTP was largely independent of [Ca2+]o. Both authors concluded that accumulation of 'active Ca' inside the nerve terminals during the tetanus underlay PTP. If Li results in less of the Ca that enters during a tetanus being

TRANSMITTER RELEASE AND Li+ 137 sequestered then it might increase the amount of 'active Ca' present to cause PTP. One striking feature of the increases in transmitter release that occur in Li-Ringer is that changes in evoked release are predictably 'locked' to changes in spontaneous release. The m.e.p.p. frequency rises exponentially with the same time constant as that of the exponential rise in the extra quantal content of the e.p.p. It is not clear why the increases should be exponential in the first place or why quantal release in the two release modes should be locked in such a fashion. However, it is possible to put forward a tentative explanation for these results on the basis of a co-operative action of Ca2+ ions in the release of a quantum of transmitter (Dodge & Rahamimoff, 1967) providing that the initial assumption is made that the free Ca concentration in the terminals, [Ca2+]R, rises exponentially with time during a Li exposure. 1-0

4j

0

4

05

A

0

J

log [Ca2+] Fig. 20. Schematic representation of the action of an intracellular Ca buffer (M). The Figure shows the titration curve. Ordinate: ratio of the total Ca concentration [Ca2+]t~t to the total buffer concentration, [M2+]tt. Abscissa: free Ca concentration at equilibrium, [Ca2+]. The resting titration state of the buffer is indicated by the point A. See text for further

explanation. If Li leaks into the terminals at a constant rate (or more realistically, progressively more slowly as the [Li+], rises) the exponential nature of the increases in transmitter release is puzzling. One simple explanation arises if the nerve terminals contain a Ca buffer with equilibrium properties similar to that of EGTA, i.e. one that binds Ca2+ but not Mg2+ with the same equilibrium constants as EGTA, but with rate constants that are very much slower. Detailed descriptions of the calcium-binding p-operties of nerve axoplasm have been published (Blaustein & Hodgkin, 1969; Baker et al. 1971) and the idea of an EGTA-like buffer is only a simplified version of these. In Fig. 20 the titration curve for the pi esynaptic buffer, M, is schematically illustrated. The essential feature is that a plot of the ratio of the total buffer concentration, [M]T to the total calcium concentration, [Ca]T, against the free calcium

138

A. C. CRAWFORD

concentration at equilibrium, [Ca2+] on semilogarithmic co-ordinates, has a linear portion over the effective buffering range. For tetra-acetic acid substituted diamino compounds such as EDTA and EGTA the effective buffering range spans two orders of magnitude change in [Ca2+]. In a normal end-plate [M]T would be fixed and [Ca]T determined by those processes responsible for the long term Ca balance of the cell - p -obably the Ca transport mechanisms of the external membrane. At the beginning of a lithium exposure the titration state of the buffer is placed at some arbitrary point A on the curve. It seems reasonable to suggest that the resting [Ca]T/[M]T would be low (< 0.5) since this would allow the buffer to accommodate large entries of Ca2+ and nerve terminals are unlikely to be subjected to a fall in [Ca]T since excitable tissues are far from electrochemical equilibrium with respect to calcium (Keynes & Lewis, 1956; Hodgkin & Keynes, 1957; Ashley & Ridgway, 1970; Baker, 1972). If a constant leak of Li into the terminals inactivates the buffer such that effectively the [M]T falls linearly with time then the titration state of the terminals will move up the buffer curve from point A in Fig. 20 to higher values of [Ca2+]. Hence the [Ca2+] will rise exponentially with a time constant determined hy the rate of Li entry into the terminals, and by the slope of the buffer titration curve.

A model of evoked release has been suggested by Katz & Miledi (1968) and amplified by Miledi & Thies (1971) and Barrett & Stevens (1972) where the mean quantal content of the e.p.p. is approximately proportioned to the free [Ca2+], inside the terminals after an action potential. If A Ca is the change in [Ca2+], produced by entry during the action potential and m = K1 ([Ca2+]R+A Ca)4. K1 a constant, then

Thus if [Ca2+]R is rising exponentially during a Li exposure the mean quantal content at time t, (mt) will be given by mt = K1 ([Ca2+]R eti? + A Ca) 4, t = 0, where r is the time constant of the rise in [Ca2+]R. Provided that A Ca is large compared to [Ca2+]R at all times it can be shown that Mt-M = 4K1 [Ca2+]R . A Ca3 (etIT -1), t = 0, where mO is the value of m at zero time. This is a reasonable description of the time course of changes in m during a Li exposure, i.e. Mt -MO = A (etr- 1), where A is a constant. In deriving an expression for the rise in the m.e.p.p. frequency it is not known how many Ca2+ ions act co-operatively in the spontaneous release of a quantum. Miledi & Thies (1971) have assumed that the number is 4 but to leave the question open it seems reasonable to describe the m.e.p.p. frequency by

f

K2 ([Ca2+]R)n,

TRANSMITTER RELEASE AND Li+ 139 where n Ca2+ ions co-operate in the release of a quanta and K2 is a constant. If fg and ft are m.e.p.p. frequencies at zero time and time t respectively, spontaneous release during a lithium exposure should be described by ft = fo. entry One further point that emerges from this analysis is that if 'co-operativity' does underlie the locking of evoked to spontaneous release, the time constant of the rise in m (aTm) should be equal to that of the rise in [Ca2+]R and that of the rise in the m.e.p.p. frequency (ir) should occur with a time constant 1/nths of this. Empirically 'm is equal to Tr which suggests that n = 1, a proposal that is consistent with the linearization of the Ca dependence of m at low [Ca2+]o (Crawford, 1974). However, until direct evidence is available on the behaviour of [Ca2+]R during a Li exposure it would be unproductive to pursue this hypothesis further. The author is indebted to Dr P. F. Baker for many helpful discussions during the of this work, and to the M.R.C. for financial support.

course

Note added in proof. Since this paper was submitted Balnave & Gage (1974, J. Physiol. 239, 657-675) have reported increases in the mean quantal content in Li-Ringer. These authors also cite the work of Carmody & Gage (1973, Brain Res. 50, 476-479) who find increases in the m.e.p.p. frequency caused by Li similar to some of the results presented here. REFERENCES

ASHLEY, C. C. & RIDGWAY, E. B. (1970). On the relationship between membrane potential, calcium transient and tension in barnacle single muscle fibres. J. Physiol. 209, 105-130. BAKER, P. F. (1972). Transport and metabolism of calcium ions in nerve. Prog. Biophys. molec. Biol. 24, 179-223. BAKER, P. F., HODGKIN, A. L. & RIDGtvAY, E. B. (1 97 1). Depolarization and calcium entry in squid giant axons. J. Phy8iol. 218, 709--755. BAKER, P. F., MEVEs, H. & RIDGWAY, E. B. (1973). Calcium entry in response to maintained depolarization of squid axons. J. Physiol. 231, 527-548. BALNAVE, R. J. & GAGE, P. W. (1971). Manganese ions and transmitter release. Proc. Aust. Phy&iot. Pharmacol. Soc. 2, 19. BARRETT, E. F. & STEVENS, C. F. (1972). The kinetics of transmitter release at the frog neuromuscular junction. J. Physiol. 227, 691-708. BRKs, R. I. & COHEN, M. W. (1965). Effect of sodium on transmitter release from frog motor nerve terminals. In Muscle, edited by PANT, W. M., DAVID, E. E., KAY, C. M. and MONCTON, G. Oxford: Pergamon. BIRKs, R. I. & COHEN, M W (1968a) The action of sodium pump inhibitors on neuromuscular transmission Proc. R. Soc. B 170, 381-399. BIRKS, R. I. & COHEN, M. W. (1968b). The influence of internal sodium on the behaviour of motor nerve endings. Proc. R. Soc. B 170, 401-421. BLAUSTEIN, M. P. & HODGKIN, A. L. (1969). The effect of cyanide on the efflux of calcium from squid axons. J. Physiol. 200, 497-529.

140 A. C. CRAWFORD BLoCH, L., GLAGOLEVA, I. M., LIBERMAN, E. A. & NENAsiEv, V. A. (1968). A study of the mechanism of quantal transmitter release at a chemical synapse. J. Phy.~iol. 199, 11-37. BLIOCH, L. & LIBERMAN, E. A. (1970). The influence of beryllium ions on the endplate potential and frequency of miniature end-plate potentials. Biofizilca 15, 468-474.

CARMELTIET, E. (1964). Influence of lithium ions

on the transmembrane potential and cation content of cardiac cells. J. gen. Physiol. 47, 501-530. CHANDLER, W. K. & MEVES, H. (1965). Voltage clamp experiments on internally perfused giant axons. J. Physiol. 180, 788-820. CLARK, A. W., HURLBUT, W. P. & MAtnRO, A. (1972). Changes in the fine structure of the neuromuscular junction of the frog caused by black widow spider venom. J. cell Biol. 52, 1-14. COLOMBO, F. & RAHAMIMOFF, R. (1968). Interaction between sodium and calcium ions in the process of transmitter release at the neuromuscular junction. J. Phy~siol. 198, 203-219. CooKiE, J. D., OKWAMOTO, K. & QUASTEL, D. M. J. (1973). The role of calcium in depolarization-secretion coupling at the motor nerve terminal. J. Phyaiol. 228, 459-497CRAWFORD, A. C. (1974). The dependence of evoked transmitter release on external calcium ions at very low mean quantal contents. J. Physiol. 240, 255-278. DEL CASTILLO, J. & KATz, B. (1954). Quantal components of the end-plate potential. J. Physiol. 124, 560-573. DODGE, F. A., MILEDI, R. & RAHAMIMOFF, R. (1969). Strontium and quantal release of transmitter at the neuromuscular junction. J. Physiol. 200, 267-283. DODGE, F. A. & RAHAMIMOFF, R. (1967). Co-operative action of calcium ions in transmitter release at the frog neuromuscular junction. J. Physiol. 193, 419-433. FATT, P. (1950). The electromotive action of acetylcholine at the motor end-plate. J. Physiol. 111, 408-422. FATT, P. & KATZ, B. (1952). Spontaneous subthreshold activity at motor nerve endings. J. Physiol. 117, 109-128. FLYNN, F. & MAIZELS, M. (1950). Cation control in human erythrocytes. J. Physiol. 110, 301-318. GAGE, P. W. & QUASTEL, D. M. J. (1966). Competition between sodium and calcium ions in transmitter release at a mammalian neuromuscular junction. J. Physgiol. 185, 95-123. HIEUSER, J., KATZ, B. & MiLEDI, R. (1971). Structural and functional changes at frog neuromuscular junctions in high calcium solutions. Proc. R. Soc. B 178, 407-415. HlEUSER, J. & MILEDI, R. (197 1). Effect of lanthanum ions on function and structure of frog neuromuscular junctions. Proc. R. Soc. B 179, 247-260. HODGKIN, A. L. & KATZ, B. (1949). The effect of sodium ions on the electrical activity of the giant axon of the squid. J. Phys-iol. 108, 37-77. HODGKIN', A. L. & KEYNES, B. D. (1957). Movement of labelled calcium in squid giant axons. J. Physiol. 138, 253-281. HUBBARD, J. I. (1963). Repetitive stimulation at the mammalian neuromuscular junction and the mobilization of transmitter. J. Physiol. 169, 641-662. HUBBARD, J. I. (1973). The microphysiology of vertebrate neuromuscular transmission. Physiol. Rev. 53, 674-723. HUBBARD, J. I., JoNESs, S. F. & LANDAU, E. M. (1968 a). On the mechanism by which calcium and magnesium affect spontaneous release of transmitter from mammalian motor nerve terminals. J. Physiol. 194, 381-407.

TRANSMITTER RELEASE AND Li+

141

HUBBARD, J. I., JoNEs, S. F. & LANDAU, E. M. (1968b). On the mechanism by which calcium and magnesium affect the release of transmitter by nerve impulses. J. Physiol. 196, 75-86. HUBBARD, J. I. & WILLIS, W. D. (1968). The effects of depolarization of motor nerve terminals upon the release of transmitter by nerve impulses. J. Physiol. 194, 381-405. HUTTER, 0. F. & NOBLE, D. (1960). The chloride conductance of frog skeletal muscle. J. Physiol. 151, 89-102. HU=TR, 0. F. & WARNER, A. E. (1967). The pH sensitivity of the chloride conductance of frog skeletal muscle. J. Physiol. 189, 403-425. HuxLEY, A. F. & STAMPFLI, R. (1951). Effect of potassium and sodium on resting and action potentials of single myelinated nerve fibres. J. Physiol. 112, 496-508. KAJIMOTO, N. & PIRPEKAR, S. M. (1972). Effects of manganese and lanthanum on spontaneous release of acetylcholine at frog motor nerve terminals. Nature New Biol. 235, 29-30. KATZ, B. & MILEDI, R. (1965). Propagation of electrical activity in motor nerve terminals. Proc. B. Soc. B 161, 453-482. KATZ, B. & MILEDI, R. (1968). The role of calcium in neuromuscular facilitation. J. Physiol. 195, 481-492. KATZ, B. & MILEDI, R. (1969). The effect of divalent cations on transmission in the squid giant synapse. Publ. Staz. zool. Napoli 37, 303-310. KELLY, J. S. (1965). Antagonism between Na+ and Ca2+ at the neuromuscular junction. Nature, Lond. 205, 296-297. KELLY, J. S. (1968). The antagonism of Ca2+ by Na+ and other monovalent ions at the frog neuromuscular junction. Q. JZ exp. Physiol. 53, 239-249. KEYNES, R. D. & LEWIS, P. R. (1956). The intracellular calcium contents of some invertebrate nerves. J. Physiol. 134, 399-407. KEYNES, R. D. & SWAN, R. C. (1959). The effects of external sodium concentration on the sodium fluxes in frog skeletal muscle. J. Physiol. 147, 591-625. LEHNINGER, A. L., CAm.AFom, E. & Rossi, C. S. (1967). Energy-linked ion movements

in mitochondrial systems. Adv. Enzymol. 29, 259-320. MAGLEBY, K. L. (1973). The effect of tetanic and post-tetanic potentiation on facilitation of transmitter release at the frog neuromuscular junction. J. Physiol. 234, 353-321. MALLART, A. & MARTIN, A. R. (1968). The relation between quantum content and facilitation at the neuromuscular junction of the frog. J. Physiol. 196, 593-604. MARTIN, A. R. (1955). A further study of the statistical composition of the end-plate potential. J. Physiol. 130, 114-122. MCMAHAN, V. J., SPITZER, N. C. & PEPER, K. (1972). Visual identification of nerve terminals in living isolated skeletal muscle. Proc. R. Soc. B 181, 421-430. MEIRI, U. & RAHAMIMOFF, R. (1971). Activation of transmitter release by strontium and calcium ions at the neuromuscular junction. J. Physiol. 215, 709-726. MEmI, U. & RAHAMIMOFF, R. (1972). Neuromuscular transmission: inhibition by manganese ions. Science, N. Y. 176, 308-309. MILEDI, R. & THIEs, R. E. (1971). Tetanic and post-tetanic rise in the frequency of miniature end-plate potentials in low calcium solutions. J. Physiol. 212, 245-257. NIEDERGERKE, R. & ORKAND, R. K. (1966). The dependence of the action potential of the frog's heart on the external and intracellular sodium concentration. J. Physiol. 184, 312-334. ONODERA, K. & YAMAKAWA, K. (1966). The effects of lithium on the neuromuscular junction of the frog. Jap. J. Physiol. 16, 541-550.

142

A. C. CRAWFORD

OVERTON, E. (1902). Beitrdge zur allgemeinen Muskel-und Nervenphysiologie. Pfluiger8 Arch. ges. Physiol. 92, 346-386. RAHAMIMOFF, R. (1968). A dual effect of calcium ions on neuromuscular facilitation. J. Physiol. 195, 471-481. RAHAMIMOFF, R. & YAARI, Y. (1973). Delayed release of transmitter at the frog neuromuscular junction. J. Physiol. 229, 241-257. RosENTn1AL, J. (1969). Post-tetanic potentiation at the neuromuscular junction of the frog. J. Physiol. 203, 121-133. SITLEN, L. G. & MARTELL, A. E. (1964). Stability Constants of Metal Ion Complexes. London: Chemical Society. TAxEUcHI, A. & TAK uciI, N. (1960). On the permeability of the end-plate membrane during the action of the transmitter. J. Physiol. 154, 52-67. WEINREICH, D. (1971). Ionic mechanisms of post-tetanic potentiation at the neuromuscular junction of the frog. J. Physiol. 212, 431-446.