J. Physiol. (1975), 248, pp. 285-306 With 12 text-ftyure8 Printed in Great Britain
285
ON THE ROLE OF MITOCHONDRIA IN TRANSMITTER RELEASE FROM MOTOR NERVE TERMINALS
BY E. ALNAES* AND R. RAHAMIMOFFt Department of Neurobiology, Harvard Medical School, Boston, Mass. 02115, U.S.A.
(Received 19 July 1974) SUMMARY
1. The changes in transmitter release produced by mitochondrial inhibitors has been studied at the frog neuromuscular junction using conventional electrophysiological techniques for stimulation and intracellular recording. 2. Inhibitors of the electron transport chain and inhibitors of oxidative phosphorylation produce an increase in the frequency of appearance of the miniature end-plate potentials. This increase in frequency is observed also in calcium-free media. Mitochondrial inhibitors also augment the amount of transmitter liberated by a nerve impulse. 3. Ruthenium red, which is an inhibitor of calcium uptake by mitochondria, increases the spontaneous transmitter release but decreases the quantal content. The latter effect of Ruthenium red is antagonized by calcium. 4. The mitochondrial content of the motor nerve terminals is, on the average, 6-59% 5. The experimental results are explained on the hypothesis that spontaneous release of transmitter reflects the resting level of intracellular free calcium and the evoked release reflects the sum of the resting calcium and the calcium brought in by the action potential. The mitochondria play a role in transmitter release by participating in the regulation of the intracellular free Ca. INTRODUCTION
Neurotransmitter is liberated from motor nerve terminals as preformed packets, or quanta (Fatt & Katz, 1951; Katz, 1969). At rest these quanta appear at the post-synaptic membrane as spontaneously occurring * Present address: Institute of Neurophysiology, University of Oslo, Oslo, Norway.
t Present address: Department of Physiology, Hebrew University-Hadassah Medical School, P.O. Box 1172, Jerusalem, 91000, Israel.
E. ALNAES AND R. RAHAMIMOFF miniature end-plate potentials (m.e.p.p.s). After the arrival of the nerve action potential at the terminals, the rate of discharge of these quanta is increased by several orders of magnitude; hundreds of quanta are released nearly simultaneously (Katz & Miledi, 1965), producing the end plate potential (e.p.p.). This increase in the rate of release upon depolarization is highly dependent on the extracellular calcium concentration (del Castillo & Stark, 1952; del Castillo & Katz, 1954a, b, c; Jenkinson, 1957; Dodge & Rahamimoff, 1967; Hubbard, Jones & Landau, 1968). Upon depolarization, calcium conductance of the terminal axon membrane increases and a calcium influx occurs. This voltage dependent calcium conductance is probably the link between the extracellular calcium concentration and transmitter release (Katz & Miledi, 1967, 1969; Baker, Hodgkin & Ridgway, 1971; Baker, 1972). A number of agents known to interfere with calcium entry also inhibit transmitter release (see Baker, 1972). These observations suggest that the intracellular calcium concentration near the presynaptic membrane may determine the amount of transmitter liberated. Such a suggestion is supported by the observation that direct intracellular application of calcium in the giant synapse of the squid increased the background rate of transmitter release (Miledi, 1973). Several processes that affect the intracellular calcium concentration might thus also change transmitter release. Thus, the amount of free intracellular calcium would conceivably be determined by the balance of several processes, such as influx, efflux and the 'buffering action' of intracellular organelles (see Fig. 11 in the Discussion). One of the likely candidates for such intracellular removal and supply of calcium ions is the mitochondrion. In a number of tissues, the mitochondria are able to take up calcium against large concentration gradients (see Lehninger, 1970). It was the purpose of this work to examine the possibility that mitochondria can affect transmitter release by regulating the intracellular calcium concentration. Some of the results presented here have been published elsewhere in brief form (Rahamimoff & Alnaes, 1973; Alnaes, Meiri, Rahamimoff & Rahamimoff, 1974). 286
METHODS Experiments were done at room temperature (2024° C) on the cutaneopectoris muscle of the frog (Rana pipiens). Muscle fibres were impaled by potassium chloride micro-electrodes (20-80 £2 resistance), 50-200 /tm from their end-plates which were viewed with Nomarski interference optics. To avoid contraction upon nerve stimulation, the preparation was bathed in a high magnesium, Ringer solution of the following composition: 103 mM-NaCl, 2 mM-KCl, 1-8 mM-CaCl2, 12 nM.Mgel2, 1 mM-Na2HPO4. The pH of the solution varied between 6-8 and 7-2.
MITOCHONDRIA AND TRANSMITTER RELEASE
287
The perfusion fluid could be changed in less than 1 min without excessive fluid turbulence and changes in muscle fibre membrane potentials. The nerve was stimulated by 0-1 msec pulses through a suction electrode, usually at a rate of 0-5 see-', and end-plate potential responses were photographed continuously on moving film. The following chemicals were used: Dicoumarol (Endo Laboratories, Inc.), Guanidine (Sigma Chemical Co.), Rotenone (K & K Laboratories, Inc.) Antimycin (Calbiochem), and Ruthenium Red (Sigma Chemical Co.). The commercial product contains approximately 20 % of the dye, the rest being NaCl. The concentrations were calculated accordingly. The mitochondrial fraction of nerve terminal volume was estimated by cutting and weighing profiles of 78 cross-section electromicrographs of two neuromuscular junctions. The areas of the mitochondrial and the nerve terminal cross-sections were measured separately, and the terminal volume calculated knowing the total length of the unmyelinated part of the nerve-terminal arborizations in the endplates from previous light micrographic measurements. RESULTS
The effect of non-8pecific mitochondrial inhibitors on transmitter release Mitochondria are able to take up calcium ions from their environment by a number of processes (Carafoli & Rossi, 1971; Carafoli, Gazzotti, Rossi & Tiozzo, 1971). Two of them, namely the binding to the high as well as low affinity sites, do not need metabolic energy. In addition, there is an energy dependent uptake process for calcium (see Lehninger, 1970). When the process of energy production is inhibited, not only is the calcium uptake ability of the mitochondria greatly reduced, but they may even lose the already sequestrated calcium. The resulting increase in intracellular [Ca] ([Ca]1n) would be expected to raise the level of spontaneous transmitter release. If one examines previous experiments on nerve terminals deprived of their energy supply, the frequency of the spontaneous m.e.p.p.s. was increased in all cases (Kraatz & Trautwein, 1957; Hubbard & L0yning, 1966; Glagoleva, Liberman & Khashayev, 1970; Katz & Edwards, 1973). An illustration of such a phenomenon is given in Fig. 1. Several minutes after the addition to the perfusing fluid, of a dicoumarol (sodium warfarin), which uncouples oxidative phosphorylation, the frequency of the spontaneous m.e.p.p.s. increased more than tenfold. Although the effect of dicoumarol on m.e.p.p. frequency is clear cut, the interpretation of this result is not. The uptake of calcium by mitochondria is not the only energy requiring mechanism in the presynaptic nerve terminal. Among other processes, the maintenance of the resting potential also requires energy. Hence, it is possible that the increase in m.e.p.p. frequency after dicoumarol is due to membrane depolarization of the terminals, which is known to lead to a very substantial increase in
288Q!
E. ALNAES AND R. RAHAMIMOFF
A
B
L Fig. 1. Effect of dicoumarol on m.e.p.p. frequency. A: control. B: after application of 2-5 mm dicoumarol in perfusing solution. Calibration bars: 1 mV, 20 msec.
m.e.p.p. frequency (del Castillo & Katz, 1954c). Depolarization of the terminals cannot be measured at the frog neuromuscular junction. However, indirect evidence suggests that this is not the primary mode of action of dicoumarol. Depolarization induced release depends to a large extent on the presence of calcium ions in the bathing medium. In the absence of external calcium ions, depolarization has only a small effect on m.e.p.p. frequency. If, on the other hand, the increase in spontaneous release is mainly secondary to calcium leakage from the mitochondria, the presence or the absence of extracellular calcium would have no great influence. Fig. 2 shows that dicoumarol increases the rate of the spontaneous events even in the absence of extracellular calcium.
MITOCHONDRIA AND TRANSMITTER RELEASE
289
30 r
20 I0
10 00 00 00
u
4
V)
0
3 .
21_ a
iI*
b
~*00S.00 * @0.0%
I.. 0000 _
*
0
0 -
P9%
*
0.0.
.
I
10
20
|
30 Min
C.|
40
50
60
Fig. 2. Dicoumarol effect on m.e.p.p. frequency in calcium-free Ringer solution. a: change of control solution. b: addition of 250 pI dicoumarol. c: addition of 2-5 mM dicoumarol.
Effect of dicoumarol on evoked quantal release Another way of excluding the possibility that the main effect of dicoumarol on spontaneous m.e.p.p. frequency is due to presynaptic depolarization is to examine its effect on evoked release. It has been shown directly at the squid giant synapse (Katz & Miledi, 1967 b) and indirectly at the vertebrate neuromuscular junction (Hubbard & Willis, 1962), that after depolarization the action potential is less effective in inducing transmitter release. If dicoumarol exerts its effect on m.e.p.p. frequency by depolarization, then it would be expected that the evoked transmitter release would be diminished after dicoumarol. If, on the other hand, the action of dicoumarol results from leakage of calcium from the
290 E. ALNAES AND R. RAHAMIMOFF mitochondria in the nerve terminal, this calcium will add to the calcium entering during the action potential; this would lead to an increase in the total internal [Ca] and potentiation in evoked release. Figs. 3 and 4 show that after moderate dicoumarol poisoning there is an increase in the quantal content, and consequently in the end-plate potential amplitude. The e.p.p. amplitude sometimes can be suprathreshold and lead to contraction (Fig. 3). The increase in quantal content, following dicoumarol is relatively short lived. After about j-1 hr (depending on B
A
C
4' msec Fig. 3. Effect of dicoumarol on evoked transmitter release. A: control series of e.p.p.s in high magnesium low calcium Ringer solution. B: 7 min after addition of 809W dicoumarol to perfusate. C: 20 min after addition. Muscle fibres in the vicinity of the impaled fibre now contract upon nerve stimulation, due to the increase in their e.p.p.s (note contraction artifact in two of the traces).
dicoumarol concentration) there is a failure in e.p.p. formation, presumably as a result of blockage of invasion of the motor nerve terminals by the action potential (cf. Krnjevi6 & Miledi, 1959; Hubbard & L0yning, 1966). Fig. 4 shows graphically the increase in m.e.p.p. frequency and in quantal content following the addition of 80 gM dicoumarol. Dicoumarol is known to be a very potent inhibitor of mitochondrial metabolism. We also tested other mitochondrial inhibitors. The action of other mitochondrial inhibitors on transmitter release Guanidine facilitates myoneural transmission and it has been used clinically to improve depressed neuromuscular transmission (Feng, 1938; Minot, Dodd & Riven, 1938; Otsuka & Endo, 1960; Cherington & Ryan,
MITOCHONDRIA AND TRANSMITTER RELEASE 291 1968; Scaer, Tooker & Cherington, 1969). Guanidine has also been shown to have an inhibitory effect on mitochondria (Pressman, 1963; Chance & Hollunger, 1963). Fig. 5 illustrates that guanidine increases the quantal content of the e.p.p. and the frequency of the spontaneous m.e.p.p. Not only uncoupling agents augment transmitter release. Similar observations were made using mitochondrial inhibitors that act at the electron transport chain level (see Fig. 12 in the Discussion). Thus rotenone (20 /tM) and antimycin (200 /TM) increase the frequency of the spontaneous m.e.p.p., that becomes more pronounced after nerve stimulation (Fig. 6). 15
m
10~~~~~~~~~~~~ 10
.
0*
*
.*.-~~~~5
.5
*
..*.@-.**. .... f 1
0
11o
..
00
5
10
15
20
Fig. 4. Dicoumarol effect on spontaneous and evoked transmitter release. Each point denotes the average quantal content (m) of thirty consecutive nerve stimulations in high magnesium (10 mM) low calcium (0.7 mM) solution. Lower curve shows corresponding changes in the frequency of miniature end-plate potentials (right-hand ordinate). Arrow marks addition of 80 /ZM dicoumarol.
Ruthenium red and release of transmitter All inhibitors of calcium uptake that have been discussed so far work through a rather generalized energy deprivation of mitochondria. They inhibit a number of energy requiring processes in the mitochondria, such as calcium uptake, ATP production and others. To focus more specificially on calcium uptake by mitochondria, we used the inorganic dye Ruthenium red (RuR) which inhibits both passive and active calcium entry in mitochondria (Moore, 1971; Vasington, Gazotti, Tiozzo & Carafoli, 1972). RuR is widely used for histochemical staining, mainly of mucopolysaccharides (see Luft, 1971 a, b). Since RuR penetrates nerve membranes, presumably through their unmyelinated segments (Singer, Krishnan &
292 E. ALNAES AND R. RAHAMIMOFF Fyfe, 1972) we examined its effect on both spontaneous and evoked transmitter release. Two main questions were posed: (1) does RuR, by blocking calcium transport into nerve mitochondria, cause an increase in spontaneous miniature e.p.p. frequency; (2) if the sole effect of RuR is on calcium transport in mitochondria, one would expect an augmentation of evoked release, similar to that produced by non-specific mitochondrial blocking agents. On the other hand, if the calcium transport process across the surface membrane of the nerve (which presumably is responsible for evoked release), shares common properties with the calcium transport process into the mitochondria, RuR will suppress evoked release. B
A
C
ms
0.0 mm-G
20 mM -G
so mm -G
Fig. 5. Effect of guanidine on evoked and spontaneous transmitter release. A: control series in high magnesium (10 mM) low calcium (0.7 mM) Ringer solution. B: after addition of 2 mm guanidine (G). C: after addition of 5 mM guanidine. Note increase in frequency of spontaneous miniature end-plate potentials, as well as in amplitude and quantal content of evoked end-plate potentials.
Effect of RuR on spontaneous release On examining the effect of RuR on transmitter release, a depressing action of RuR on the amplitude (a) of the spontaneous miniature e.p.p.s. was observed. This inhibitory effect increased with the RuR concentration (Fig. 7), and was observed in 11 other preparations. In two experiments done in 40,uM-RuR, any synaptic activity was undistinguishable from
MITOCHONDRIA AND TRANSMITTER RELEASE A
8
C
D
E
F
293
40 msec 2
m
V
Fig. 6. Effect of antimycin on transmitter release. A: control series of stimulations in high magnesium low calcium Ringer solution. B: immediately after addition of 05 mm antimycin to extracellular fluid. C: 1 min later, at 0-5 see'. D: double stimulation at 1 0 sec1 which brings about a very large increase in m.e.p.p. frequency. E: immediately after resuming single stimulation at original low rate (05 sec"). F: 2 min after resuming control stimulation parameters. Quantal content in the control situation was 1 1. After 15 min in antimycin, the quantal content was 1D95.
nerve
E. ALNAES AND R. RAHAMIMOFF the noise. This presumed post-synatpic effect of RuR imposed an upper limit on the useful concentration ranges. Reduced amplitude of the m.e.p.p. can arise from an increase in the membrane conductance of the muscle fibre (Katz & Thesleff, 1957). Such an explanation does not apply to the action of RuR, since in two experiments it was found that the input resistance of the post-synaptic muscle fibre increased upon addition of RuR. 294
0-8 F
06 F
0 i
(MY)
.
04
0*2 .
Ulum
0
*
II
2-5
5-0
7.5
PM
Fig. 7. Post-synaptic effect of Ruthenium red (RuR). Average miniature end-plate potential amplitude a measured at the same neuromuscular junction at three different RuR concentrations.
The effect of RuR (5 pM) on m.e.p.p. frequency was examined in eight experiments in which the initial amplitude of the spontaneous events was large enough so that, even after the depression caused by RuR, the m.e.p.p.s would still be clearly distinguishable from the noise. It was found that on the average there was approximately a doubling in m.e.p.p. frequency after 15-20 min exposure to RuR (range 146-400 % of the control).
MITOCHONDRIA AND TRANSMITTER RELEASE
295
Evoked transmitter release and ruthenium red RuR has a very strong inhibitory effect on e.p.p. amplitude. Addition of 5-10 /LM-RuR to normal Ringer, with 1'8 mm calcium, is usually sufficient to block normal transmission across the neuromuscular synapse and prevent contractions. Analysis of this action shows that the main effect of RuR is on evoked transmitter release; the quantal content is B
A
2 mV
40 msec
Fig. 8. Effect of Ruthenium red (RuR) on evoked and spontaneous transmitter release. A: control series of nerve stimulations in high magnesium low calcium-Ringer solution. B: 10 min after addition of 5 /SM-RuR to extracellular fluid. Observe decrease in quantal content and m.e.p.p. amplitude, as well as increase in m.e.p.p. frequency.
very substantially decreased after RuR (Fig. 8). The effect of RuR in reducing evoked release of transmitter was seen in seventeen preparations under various experimental conditions. A prolonged wash in RuR-free medium only partially restores transmitter liberation. The inhibitory action of RuR on evoked release of transmitter does not arise from inability of the terminals to liberate quanta of acetylcholine
E. ALNAES AND R. RAHAMIMOFF 296 upon the arrival of the nerve impulse. This can be shown by increasing the calcium concentration in the external medium. Fig. 9 illustrates such an experiment. Neuromuscular transmission was blocked by addition of 1OpM-RuR to standard Ringer (1.8 mm calcium) and the resulting subthreshold e.p.p. amplitude was on the average 2 mV. Doubling [Ca] (at the first bar) and quadrupling it (at the second bar) produced a very substantial increase in e.p.p. amplitude and quantal 12
10 8 -~~~~~~~~~:
8 mV 6
4.
2
m
_
0
12 10 8 6 Min Fig. 9. Calcium-induced increase in e.p.p. amplitude (and quantal content) when neuromuscular transmission has been suppressed by 10 /laM Ruthenium red. Extracellular Ca-concentration doubled successively at the two filled bars. Each point represents the mean amplitude of ten responses. See text for further details. 0
2
4
content (the quantal contents at the steady-state level were 21-7, 42-5, and 112 respectively, estimated from the variance; cf. Martin 1955). If the concentration of calcium in the extracellular medium was increased to 14.4 mm (8 times the standard), evoked release rates reached values of about 250 (257.6) in the presence of RuR, which are in the normal range of release (Katz, 1962). However, the e.p.p. amplitudes were still subthreshold, due to the post-synaptic inhibitory action of RuR.
MITOCHONDRIA AND TRANSMITTER RELEASE
297
A
0-4
0
03 0 as
0
0
pm2 02[
0 0
0
0
.
01 _
. * *
0 0 0 0~
*
.
0
U *
0
.
0
0
*0He
*0 C0 'moe
*Uh
U-
40r
*
0
* -
a
I*
I
5
-2
I
__j
I
2
3 pm2
4
5
6
B 0
30 0.
*
a
0
*@-
U *
0
0
10
0~~~~ 0
No .
EU 0
.
0
*
*
0
- I AV
a
I
0
*
A l_ Vi
*"*
0
-
I
'*
m
* -
I |
I __
I
6 5 3 4 PM2 Fig. 1OA. Mitochondrial cross-sectional area-Am (ordinate) plotted against nerve terminal area-An (abscissa) in seventy-eight cross-sections of unmyelinated portion of presynaptic nerve terminals in two different muscles (squares and circles). B: mitochondrial cross-sectional area (ordinate) plotted as percentage of nerve terminal area (abscissa). Same data as in A. 0
1
2
298
E. ALNAES AND R. RAHAMIMOFF
Mitochondrial content of nerve terminal The presence of large numbers of mitochondria in nerve terminals has long been recognized (see Birks, Huxley & Katz, 1960). To obtain quantitative information we used seventy-eight electron micrographs taken from random sections through nerve terminals of two muscles. The micrographs were kindly provided by Mr Kenneth Fischbeck. The terminals were enlarged 63000 x, cut out of the electron microscopic photographs and the paper was weighed. From the 'weight' of 1 /um2, in the photographs, one can convert the 'weights' of the terminal profiles into cross-sectional areas. A similar procedure was repeated for the mitochondria. The results are presented in Fig. 10 in two ways. In Fig. 10A the area of all the mitochondria in a given section (Am) is plotted against area of the nerve terminal cross-section (A.). The number of mitochondria in Am varied between 0 and 8. Fig. 10B is based on the same data as 10A, but shows the fractional occupation by the mitochondria, the ordinate being (Am/An) x 100. There is a considerable variation in the mitochondrial content of nerve terminal sections, the range being between 0 and 38-6 %. On the average the mitochondria occupy 6-59 % of the nerve terminal. DISCUSSION
The basic assumption in this discussion is that the calcium concentration inside the nerve terminal controls transmitter release (see Katz & Miledi, 1967a, b). The exact location of the action of intracellular ionized calcium is still unknown, and for simplicity we have assumed that the free calcium ion concentration near the inside of the presynaptic membrane determines transmitter release. (The qualitative interpretation of the experimental results will be similar if we assume that release of transmitter depends on the surface density of calcium ions on the inside of the presynaptic membrane; which in turn will depend on the free intracellular calcium concentration.) The free intracellular calcium concentration will depend on a balance between several processes: (1) the resting calcium influx and the calcium influx following depolarization (Baker, Hodgkin & Ridgway, 1971) which raises [Ca]in; (2) the opposing calcium efflux (Baker & Blaustein, 1968; Blaustein & Hodgkin, 1969; Baker, Blaustein, Hodgkin & Steinhardt, 1969; DiPolo, 1973; Mullins & Brinley, 1975) which lowers [Ca]in; (3) intracellular calcium buffers such as the mitochondria (Lehninger, 1970); and other calcium binding systems (see Hillman & Llinas, 1974; Oschman, Hall, Peters & Wall, 1974), that are able to bind calcium reversibly. Thus, the regulation of the intracellular calcium
MITOCHONDRIA AND TRANSMITTER RELEASE 299 concentration is a complex process, depending on the contributions of the various components (see schematic representation in Fig. 11). In the present work we tried to examine the role of one of the components in this scheme, namely the mitochondria.
[CO+]
0 0
Fig. 11. Factors which influence the intracellular calcium level which in turn may determine transmitter release. See text for further discussion.
Several lines of evidence suggest that mitochondria can indeed play a part in transmitter release. Mitochondria are abundant in the nerve terminal portion of the nerve; they occupy about 6 % of the volume. This figure is significantly higher than the figure of 1 % found in the squid giant axon (DiPolo, 1973). The average length of the terminals in the data shown in Fig. 10 was approximately 700 gtm (688 + 204; mean + S.D.) and the mean cross-section P5 /Zm2. Hence, the mean volume of the terminal will be approximately 1050 ,nm3. With a value of 6 % mitochondrial mass, one obtains 63 /um3 of mitochondria in the terminal. If the specific gravity of mitochondria is about 1 and the mitochondrial mass contains about 10 % protein, and the calcium binding is 300 x 10-9 mole/mg protein, then the mitochondrial calcium binding capacity is approximately 1P9 x 10-15 mole calcium per terminal. This value exceeds by several orders of magnitude the amount of calcium that enters the nerve terminal due to an action potential; it has to be recognized, though, that the values were taken from different species, under various experimental conditions; nor do we know at present the extent of calcium binding under physiological conditions. However, there is little doubt that calcium binding capacity of the mitochondria well exceeds the influx brought about by an action potential. The response of the nerve terminal to mitochondrial inhibition is rather uniform. Regardless of the point of attack within the mitochondrial metabolism, the terminals increase their level of spontaneous transmitter
E. ALNAES AND 1. RAHAMIMOFF release. Thus, the same result if one inhibits the mitochondria at the electron transport chain level by rotenone, antimycin, cyanide or lack of oxygen (Hubbard & L0yning, 1966; Katz & Edwards, 1973; Alnaes, Rahamimoff, Rotshenker & Shimoni, 1974; Alnaes, Meiri, Rahamimoff & Rahamimoff, 1974) or at the coupling stage (Glagoleva, Liberman & Khashaiev, 1970; Katz & Edwards, 1973; Rahamimoff & Alnaes, 1973), namely an increase in the spontaneous release rate (Fig. 12). 300
Succinate
Glutamate NAD
____
Pyruvate
Calcium uptake
>
A \I / B
CoQ, cyt b -
72
ADP
2
Ascorbate X C D cyt c1-cyt c- cyt a: a3-
2
33
ATP
Fig. 12. Metabolic pathways of mitochondria. Electron transport proceeds along upper horizontal arrows. One (marked (1)) of three ATP generating side branches is drawn schematically. Calcium uptake is thought to occur facultatively to ADP-ATP conversion. Probable sites of action of mitochondrial inhibitors: A: barbiturates, rotenone. B: antimycin. C: cyanide. D: hypoxia. E: uncoupling agents (e.g. dicoumarol, DNP). F: specific calcium uptake antagonists (e.g. Ruthenium red) (after Lehninger, 1970; Carafoli et al. 1971; Carafoli & Rossi, 1971).
It seems simplest to assume that the increase in the spontaneous release of transmitter results from an increase in the intracellular calcium concentration in the terminals. This in turn can arise from a disturbance in the balance between influx and efflux through the presynaptic membrane, uptake by the mitochondria, calcium leakage from mitochondria or other calcium binding intracellular structures. These processes cannot be separately assessed with present methods at the neuromuscular junction. But the fact that an increase in spontaneous release of transmitter can be observed even in the absence of appreciable amounts of calcium in the extracellular fluid suggests that the main source of calcium for this elevated spontaneous release is intracellular. Not only spontaneous but also evoked release of transmitter is augmented by mitochondrial inhibitors. If evoked release is a function of the calcium influx following the action potential and represents the release due to the sum of the existing intracellular calcium plus that newly entered, then after inhibition of mitochondrial calcium uptake, this quantity will be greater, leading to augmentation of evoked release.
MITOCHONDRIA AND TRANSMITTER RELEASE 301 A highly non-linear power relation has been observed between the extracellular calcium concentration and evoked release of transmitter (Jenkinson, 1957; Dodge & Rahamimoff, 1967; Katz & Miledi, 1969; Hubbard, Jones & Landau, 1968). It is not clear at present whether this non-linearity represents the relation between the extracellular calcium concentration and calcium entry, or between the intracellular calcium concentration and release. In the latter case, mitochondrial inhibition and the subsequent inhibition of calcium uptake would be very effective in determining the level of transmitter release following the action potential; thus a metabolic control of synaptic transmission can be envisaged. If the main determining factor in the non-linear relation resides in the entry process, such metabolic control will be less efficient. In any case, a power function, with an exponent greater than 1, presumably exists between [Ca]jin and transmitter release: otherwise, the evoked release will not be potentiated by more than the corresponding fraction of the extra spontaneous release that occurs during the e.p.p. (Fig. 4). Two main presynaptic effects of RuR were observed, an increase in the frequency of the spontaneous miniature e.p.p.s and a decrease in the quantal number. The first observation can be explained on the basis of the known inhibitory effect of RuR on calcium uptake by the mitochondria (Moore, 1971; Vasington et al. 1972). The observed decrease in quantal content can be accounted for, if RuR inhibits the calcium transport into the nerve terminal, similarly to its inhibition of the calcium transport into the mitochondria. The increase in m.e.p.p. frequency produced by the lanthanide praseodymium, and the biphasic action on evoked release, may have a similar origin (cf. Alnaes & Rahamimoff, 1974). It should be stressed that these two calcium transport mechanisms are different in many respects. While the first one apparently operates along the electrochemical gradient (see Katz & Miledi, 1969; Baker, Hodgkin & Ridgway, 1971; Baker, 1972; Baker, Meves & Ridgway, 1971, 1973) between the outside and the inside of the nerve terminal, the second process is an energy requiring transport process. However, it has been shown that a passive binding, presumably to the high affinity sites at the mitochondrial membrane, is a necessary step in the calcium uptake by the mitochondria (Chance, 1965; Scarpa & Azzone, 1968). GomezPuyou, Marietta de Puyou, Becker & Lehninger (1972) have further shown that the effect of RuR on calcium uptake can be accounted for by its action on the glyco-protein molecules, isolated from mitochondria, which have properties similar to the high affinity binding sites. It will be of interest to see whether glycoprotein molecules participate in the calcium entry process into the nerve terminal. After the e.p.p. the probability of release of quanta of transmitter
302 E. ALNAES AND R. RAHAMIMOFF remains at an abnormally high level for several hundreds of milliseconds, and is manifested as the so-called delayed release of spontaneous miniature e.p.p.s (Miledi & Thies, 1971; Rahamimoff & Yaari, 1973) as well as an increase in the number of quanta released by a second nerve impulse (facilitation; del Castillo & Katz, 1954d). It was proposed that this increased probability of transmitter quanta is due to residual calcium (Katz & Miledi, 1965, 1968; Rahamimoff, 1968, 1973). The observation showing that calcium uptake by mitochondria is a fast process starting to operate within tens of milliseconds after exposure (cf. Chance, 1965; Chance, Salkowitz & Kovach, 1972), suggests that mitochondria may take part in the removal of this residual release process. Furthermore, while calcium and strontium are readily taken up by mitochondria, barium is not. When barium activates transmitter release (Miledi, 1966; Alnaes, Meiri, Rahamimoff & Rahamimoff, 1974) delayed release lasts for 8-20 sec, a period much longer than that observed when calcium and strontium activate release. Thus, the residual calcium may be due to at least three processes - delayed entry of calcium (Stinnakre & Tauc, 1973), efflux of calcium through the presynaptic membrane and sequestration by mitochondria. Another aspect of transmitter release in which mitochondria may be involved is the effect of osmotic pressure. It has been shown by Fatt & Katz (1952) and Furshpan (1956) that hyperosmotic media increase spontaneous transmitter release. This finding fits with the observation of Scarpa & Azzone (1968) that the calcium uptake by mitochondria decreases with increase in osmotic pressure in the incubating medium. In developing synapses, delayed release is very high (Fischbach, 1972). This seems to fit with the observation that growing cones and developing terminals have a low mitochondrial content (Tennyson, 1970; Yamada, Spooner & Wessels, 1971; Bunge, 1972), and therefore probably a low calcium-buffering capacity. All these observations suggest that mitochondria play an important part in the regulation of intracellular calcium concentration in the nerve terminal, thereby influencing transmitter liberation and synaptic transmission. Moreover, the abundance of mitochondria in some presynaptic terminals within the central nervous system (cf. Walsh, Houk, Atluri & Mugnaini, 1972), as well as their presence near the vesicles in some sensory cells, such as the chemoreceptors in the carotid body (cf. Al-Lami & Murray, 1968; Biscoe, 1971; Eyzaguirre & Nishi, 1974) and hair cells in the acoustico-lateral line system (Flock, 1965), makes one wonder whether such a regulation of neurosecretion is not of a more generalized occurrence.
MITOCHONDRIA AND TRANSMITTER RELEASE
303 We are indebted to Mr Kenneth Fishbeck for providing the EM photographs and to Dr U. J. McMahan for morphological guidance. We are very grateful to Professor S. W. Kuffler for many valuable discussions throughout the course of the work and during the preparation of the manuscript and for his hospitality. We thank Professor B. Katz for reading the manuscript and for valuable suggestions. This work was supported by N.I.H. and by the Sloan Foundation. REFERENCES
AL-L~.i, F. & Muiuu~r, R. C. (1968). Fine structure of the carotid body of normal and anoxic cats. Anat. Rec. 160, 718. AImA"s, E., MEnui, U., RATHAMIMOFF, H. & RAHAMIoFF, R. (1974). Possible role of mitochondria in transmitter release. J. Phy8iol. 241, 30 P. ALNAE5, E. & RAH moir, R. (1974). Dual action of Praseodynium (Pr+++) on transmitter release at the frog neuromuscular synapse. Nature, Lond. 247, 478479.
ALNAEs, E., RAWANornior, R., ROTSHENKER, S. & SHMOIoa, Y. (1974). Mitochondrial inhibitors and transmitter release at the frog neuromuscular junction. Proc. Israel Physiol. & Pharmacol. Soc. Al 31st meeting. I~rael J. med. Sci. (in the
Press). BAXR, P. F. (1972). Transport and metabolism of calcium ioits in nerve. Prog. Riophy8. molec. Biol. 24, 177-223. BAKER, P. F. & BLAusTEI.N, M. P. (1968). Sodium dependent uptake of calcium by crab nerve. Biochim. biophy8. Acta 150, 167. BAKER, P. F., BL~usTEiN, M. P., HODGKIN, A. L. & STEINHARDT, R. A. (1969). The influence of calcium on sodium efflux in squid axons. J. Phyeiol. 200, 431458.
BmmxR, P. F., HODGKIN, A. L. & RIDGwAY, E. B. (1971). Depolarization and calcium entry in giant squid axons. J. Phyeiol. 218, 709-755. BARER, P. F., MEv:Es, H. & RIDGwAY, E. B. (1971). Phasic entry of calcium in response to depolarization of giant axon of Loligo forbeai. J. Phy8iot. 216, 70P. BAKECR, P. F., MEvEs, H. & RIDGwAY, E. B. (1973). Effects of manganese and other agents on the calcium uptake that follows depolarization of squid axons. J. Phyeiol. 231, 51 1-526. Bmx~s, R. I., HuxL~EY, H. E. & KATz, B. (1960). The fine structure of the neuromuscular junction of the frog. J. Physiol. 150, 134-144. BiscoE, T. J. (1971). Carotid body: structure and function. Phyeiol. Rev. 51, 437-495.
BLAusTErN, M. P. & HODGKI, A. L. (1969). The effect of cyanide on the efflux of calcium from squid axons. J. Phyeiol. 200, 497-527. BUNGEM,M. B. (1972). Fine structure of nerve fibers and growth cones in isolated sympathetic neurons in culture. J. cell Biol. 56, 713-735. CAIRAoLI, E., GAzzoTTI, P., Rossi, C. S. & Tiozzo, R. (1971). Ca++ and mitochondrial membranes: evidence for specific enzymic carriers. Adv. exp. Med. & Biol. 14, 63-85. CAFA~orLi, E. & Rossi, C. 5. (1971). Calcium transport in mitochondria Adv. Cytopharmacol. 1, 209-227. CEhkNcE:, B. (1965). The energy-linked reaction of calcium with mitochondria. J. biol. Chem. 240, 2729. CHAN,-CE, B. & HoLLuNGEHR, G. (1963). Inhibition of electron and energy transfer in mitochondria: IIL The site and the mechanism of guanidine action. J. biol. Chem. 238, 432-438.
304
E. ALNAES AND R. RAHAMIMOFF
CHANCE, B., SAL.OVITZ, I. & KOVACH, A. G. (1972). Kinetics of mitochondrial flavoprotein and pyridine nucleotide in perfused heart. Am. J. Physiol. 223, 207-218. CHERINGTON, M. & RYAN, D. W. (1968). Botulism and guanidine. New Engi. J. Med. 278, 931-933. DEL CASTILLO, J. & KATZ, B. (1954a). The effect of magnesium on the activity of motor nerve endings. J. Phy8iol. 124, 553-559. DEL CASTILLO, J. & KATZ, B. (1954b). Quantal components of the end-plate potential. J. Physiol. 124, 560-573. DEL CASTILLO, J. & KATZ, B. (1954c). Statistical factors involved in neuromuscular facilitation and depression. J. Physiol. 124, 574-585. DEL CASTILLO, J. & KATZ, B. (1954d). Changes in end-plate activity produced by presynaptic polarization. J. Physiol. 124, 586-604. DEL CASTILLO, J. & STARK, L. (1952). The effect of calcium on the motor end plate potentials. J. Physiol. 116, 507-515. DODGE, F. A. & RAHAMIMOFF, R. (1967). Co-operative action of calcium ions in transmitter release at the neuromuscular junction. J. Physiol. 193, 419-432. DIPOLO, R. (1973). Calcium efflux from internally dialysed squid giant axons. J. gen. Physiol. 62, 575-589. ECCLES, R. M., L0YNING, Y. & OSHIMA, T. (1966). Effects of hypoxia on the monosynaptic reflex pathway in the cat spinal cord, J. Neurophysiol. 29, 315-332. EYZAGUIRRE, C. & NIsHI, K. (1974). Further study on mass receptor potential of carotid body chemosensors. J. Neurophysiol. 37, 156-169. FATT, P. & KATZ, B. (1951). An analysis of the end-plate potential recorded with an intracellular electrode. J. Physiol. 115, 320-370. FATT, P. & KATZ, B. (1952). Spontaneous subthreshold activity at motor nerve endings. J. Physiol. 117, 109-128. FENG, T. P. (1936). Studies on the neuromuscular junction. The universal antagonism between calcium and curarizing agencies. Chin. J. Physiol. 10, 513-528. FENG, T. P. (1938). Studies on the neuromuscular junction. X. The effects of guanidine. Chin. J. Physiol. 13, 119-140. FISCHBACH, G. B. (1972). Synapse formation between dissociated nerve and muscle cells in low density cell cultures. Devl Biol. 28, 407-429. FLOCK, A. (1965). Electronmicroscopic and electrophysiological studies on the lateral line canal organ. Acta oto-lar. 199, 90M. FURsHPAN, E. J. (1956). The effects of osmotic pressure changes on the spontaneous activity at motor nerve endings. J. Physiol. 134, 689-697. GLAGOLEVA, I. M., LIBERMAN, Y. A. & KHASHAYEV, Z. (1970). Effect of uncoupling agents of oxidation phosphorylation on the release of acetylcholine from nerve endings. Biofizika 15, 76-83. GomEz-Puyou, A., DE Puyou, MARIETTA BECKER, G. & LEHNINGER, A. (1972). An insoluble Ca++ binding factor from rat liver mitochondria. Biochem. biophys. Res. Commun. 47, 814-819. HILLMAN, D. E. & LLINAS, R. (1974). Calcium containing electron dense structures in the axons of the squid giant synapse. J. cell Biol. 61, 146-155. HUBBARD, J. I., JoNEs, S. F. & LANDAU, E. M. (1968). On the mechanism by which calcium and magnesium affect the release of transmitter by nerve impulses. J. Physiol. 196, 75-86. HUBBARD, J. I. & LoYNING, Y. (1966). The effects of hypoxia on neuromuscular transmission in a mammalian preparation. J. Physiot. 185, 205-223. HUBBARD, J. I. & WILLIS, W. D. (1962). Hyperpolarization of mammalian motor nerve terminals. J. Physiol. 163, 115-137.
MITOCHONDRIA AND TRANSMITTER RELEASE
305
JENKNSON, D. H. (1957). The nature of the antagonism between calcium and magnesium ions at the neuromuscular junction. J. Physiol. 138, 434-444. KATZ, B. (1962). The Croonian Lecture: The transmission of impulses from nerve to muscle and the subeellular unit of synaptic action. Proc. R. Soc. B 155, 455477. KATZ, B. (1969). The release of neural transmitter substances. Sherrington Lecture no. 10, pp. 60. Liverpool: Liverpool University Press. KATZ, N. L. & EDWARDS, C. (1973). Effects of metabolic inhibitors on spontaneous and neurally evoked transmitter release from frog motor nerve terminals. Proc. 26th Meeting Soc. Gen. Physiol. J. gen. Physiol. 61, 259. KATZ, B. & MIIEDI, R. (1965). The effect of calcium on acetylcholine release from motor nerve terminals. Proc. R. Soc. B 161, 496-503. KATZ, B. & MUMEDI. R. (1967a). A study of synaptic transmission in the absence of nerve impulse. J. Physiol. 192, 407-436. KATZ, B. & MILEDI, R. (1967b). The release of acetylcholine from nerve endings by graded electric pulses. Proc. R. Soc. B 167, 23-38. KATZ, B. & MILEDI, R. (1968). The role of calcium in neuromuscular facilitation. J. Physiol. 195, 481-492. KATZ, B. & MLEDI, R. (1969a). The effect of divalent cations on transmission in the squid giant synapse. Publ. Staz. zool. Napoli 37, 303-310. KATZ, B. & MILEDI, R. (1969b). Tetrodotoxin resistant electric activity in presynaptic terminals. J. Phyaiol. 203, 459-487. KATZ, B. & THESLEFF, S. (1957). On the factors which determine the amplitude of the 'miniature end-plate potential.' J. Phyaiol. 137, 267-278. KRAATZ, H. G. & TRAuTwEIN, W. (1957). Die Wirkung von 2,4-Dinitrophenol (DNP) auf die neuromuskulare Erregung subertragung. Arch. exp. Path. Pharmak. 231, 419-439. KRNJEVI6, K. & MILEDI R. (1959). Presynaptic failure of neuromuscular propagation in rats. J. Phyaiol. 149, 1-22. LEHNINGER, A. L. (1970). Mitochondria and calcium ion transport. Biochem. J. 119, 129-138. LUFT, J. H. (1971 a). Ruthenium Red and Violet I. Chemistry, purification, methods for use for electron microscopy and mechanism of action. Anat. Rec. 171, 347368.
LUFT, J. H. (1971 b). Ruthenium Red and Violet II. Fine Structural localization in animal tissues. Anat. Rec. 171, 369-416. MARTIN, A. R. (1955). A further study of the statistical composition of the endplate potential. J. Phyaiol. 130, 114-122. MILEDI, R. (1966). Strontium as a substitute for calcium in the process of transmitter release in the neuromuscular junction. Nature, Lond. 212, 1233-1234. MILEDI, R. (1973). Transmitter release induced by injection of calcium ions into nerve terminals. Proc. R. Soc. B. 183, 421-425. MILEDI, R. & TEIIs, R. E. (1971). Tetanic and post-tetanic rise in frequency of miniature end-plate potentials in low calcium solution. J. Physiol. 212, 245-257. MINOT, A. S., DODD, K. & RIVEN, S. S. (1938). The response of the myasthenic state of guanidine hydrochloride. Science, N.Y. 87, 348-350. MOORE, C. L. (1971). Specific inhibition of mitochondrial Ca++ transport by Ruthenium Red. Biochem. biophy8. Res. Common. 42, 298-305. MuLLINS, L. J. & BRINLEY, F. J. (1975). The sensitivity of calcium efflux from squid axons to changes in membrane potential. J. gen. Physiol. 63, 135-152. OscHMAN, J. L., HALL, T. A., PETERS, P. D. & WALL, B. J. (1974). Association of calcium with membranes of squid giant axon. J. cell Biol. 61, 156-165.
306
E. ALNAES AND R. RAHAMIMOFF
OTSUKA, M. & ENDo, M. (1960). The effect of guanidine on neuromuscular transmission. J. Pharnumc. exp. Ther. 128, 213-282. PREsSMAN, B. C. (1963). The effects of guanidine and alkyl guanidines on the energy transfer reaction of mitochondria. J. biol. Chem. 238, 401-409. RAHAMImoFF, R. (1968). A dual effect of calcium ions on neuromuscular facilitation. J. Physiol. 195, 471-480. RAHAMIMOFF, R. (1973). Modulation of transmitter release at the neuromuscular synapse. In The Neuro8cience8, Third Study Program, ed. Scimrrr, F. 0. & WORDEN, F. G., pp. 943-952. Cambridge, Mass. U.S.A.: MIT Press. RAHA.MOFF, R. & ALNAEs, E. (1973). Inhibitory action of Ruthenium Red on neuromuscular transmission. Proc. natn. Acad. Sci. U.S.A. 70, 3613-3616. RAHAMIMOFF, R. & YAARI, Y. (1973). Delayed release of transmitter at the frog neuromuscular junction. J. Phyaiol. 228, 241-257. SCAER, R. C., TooxER, J. & CHERINGTON, M. (1969). Effect of guanidine on the neuromuscular block of botulism. Neurology, Minneap. 19, 1107-1 1 10. SCARPA, A. & AzzoNE, G. F. (1968). Ion transport in liver mitochondria. IV. The role of surface binding on anaerobic Ca++ translocation. J. biol. Chem. 243, 5132-5138. SINGER, M., KRISHNAN, N. & FYFE, D. A. (1972). Penetration of Ruthenium Red into peripheral nerve fibers. Anat. Rec. 173, 375-390. STINNAKRE, J. & TAuc, L. (1973). Calcium influx in active Aply8ia neurons detected by injected Aequorin. Nature, New Biol. 242, 113-115. TENNYSON, V. M. (1970). The fine structure of the axon and growth cone of the dorsal root neuroblast of the rabbit embryo. J. cell Biol. 44, 62-79. VASINGTON, F. D., GAZZOT, P., Tiozzo, R. & CARAWoLI, E. (1972). The effect of Ruthenium Red of Ca++ transport and respiration in rat liver mitochondria. Biochim. biophy8. Acta 256, 43-54. WALsH, J. V., HOUK. J. C., ATLURI, R. L. & MUGNAINI, E. (1972). Synaptic transmission at single glomeruli in the Turtle cerebellum. Science, N.Y. 178, 881-883. YAMADA, K. M., SPOONER, B. S. & WESSELS, N. K. (1971). Ultrastructure and function of growth cones and axons of cultured nerve cells. J. cell Biol. 49, 614-635.
285
ON THE ROLE OF MITOCHONDRIA IN TRANSMITTER RELEASE FROM MOTOR NERVE TERMINALS
BY E. ALNAES* AND R. RAHAMIMOFFt Department of Neurobiology, Harvard Medical School, Boston, Mass. 02115, U.S.A.
(Received 19 July 1974) SUMMARY
1. The changes in transmitter release produced by mitochondrial inhibitors has been studied at the frog neuromuscular junction using conventional electrophysiological techniques for stimulation and intracellular recording. 2. Inhibitors of the electron transport chain and inhibitors of oxidative phosphorylation produce an increase in the frequency of appearance of the miniature end-plate potentials. This increase in frequency is observed also in calcium-free media. Mitochondrial inhibitors also augment the amount of transmitter liberated by a nerve impulse. 3. Ruthenium red, which is an inhibitor of calcium uptake by mitochondria, increases the spontaneous transmitter release but decreases the quantal content. The latter effect of Ruthenium red is antagonized by calcium. 4. The mitochondrial content of the motor nerve terminals is, on the average, 6-59% 5. The experimental results are explained on the hypothesis that spontaneous release of transmitter reflects the resting level of intracellular free calcium and the evoked release reflects the sum of the resting calcium and the calcium brought in by the action potential. The mitochondria play a role in transmitter release by participating in the regulation of the intracellular free Ca. INTRODUCTION
Neurotransmitter is liberated from motor nerve terminals as preformed packets, or quanta (Fatt & Katz, 1951; Katz, 1969). At rest these quanta appear at the post-synaptic membrane as spontaneously occurring * Present address: Institute of Neurophysiology, University of Oslo, Oslo, Norway.
t Present address: Department of Physiology, Hebrew University-Hadassah Medical School, P.O. Box 1172, Jerusalem, 91000, Israel.
E. ALNAES AND R. RAHAMIMOFF miniature end-plate potentials (m.e.p.p.s). After the arrival of the nerve action potential at the terminals, the rate of discharge of these quanta is increased by several orders of magnitude; hundreds of quanta are released nearly simultaneously (Katz & Miledi, 1965), producing the end plate potential (e.p.p.). This increase in the rate of release upon depolarization is highly dependent on the extracellular calcium concentration (del Castillo & Stark, 1952; del Castillo & Katz, 1954a, b, c; Jenkinson, 1957; Dodge & Rahamimoff, 1967; Hubbard, Jones & Landau, 1968). Upon depolarization, calcium conductance of the terminal axon membrane increases and a calcium influx occurs. This voltage dependent calcium conductance is probably the link between the extracellular calcium concentration and transmitter release (Katz & Miledi, 1967, 1969; Baker, Hodgkin & Ridgway, 1971; Baker, 1972). A number of agents known to interfere with calcium entry also inhibit transmitter release (see Baker, 1972). These observations suggest that the intracellular calcium concentration near the presynaptic membrane may determine the amount of transmitter liberated. Such a suggestion is supported by the observation that direct intracellular application of calcium in the giant synapse of the squid increased the background rate of transmitter release (Miledi, 1973). Several processes that affect the intracellular calcium concentration might thus also change transmitter release. Thus, the amount of free intracellular calcium would conceivably be determined by the balance of several processes, such as influx, efflux and the 'buffering action' of intracellular organelles (see Fig. 11 in the Discussion). One of the likely candidates for such intracellular removal and supply of calcium ions is the mitochondrion. In a number of tissues, the mitochondria are able to take up calcium against large concentration gradients (see Lehninger, 1970). It was the purpose of this work to examine the possibility that mitochondria can affect transmitter release by regulating the intracellular calcium concentration. Some of the results presented here have been published elsewhere in brief form (Rahamimoff & Alnaes, 1973; Alnaes, Meiri, Rahamimoff & Rahamimoff, 1974). 286
METHODS Experiments were done at room temperature (2024° C) on the cutaneopectoris muscle of the frog (Rana pipiens). Muscle fibres were impaled by potassium chloride micro-electrodes (20-80 £2 resistance), 50-200 /tm from their end-plates which were viewed with Nomarski interference optics. To avoid contraction upon nerve stimulation, the preparation was bathed in a high magnesium, Ringer solution of the following composition: 103 mM-NaCl, 2 mM-KCl, 1-8 mM-CaCl2, 12 nM.Mgel2, 1 mM-Na2HPO4. The pH of the solution varied between 6-8 and 7-2.
MITOCHONDRIA AND TRANSMITTER RELEASE
287
The perfusion fluid could be changed in less than 1 min without excessive fluid turbulence and changes in muscle fibre membrane potentials. The nerve was stimulated by 0-1 msec pulses through a suction electrode, usually at a rate of 0-5 see-', and end-plate potential responses were photographed continuously on moving film. The following chemicals were used: Dicoumarol (Endo Laboratories, Inc.), Guanidine (Sigma Chemical Co.), Rotenone (K & K Laboratories, Inc.) Antimycin (Calbiochem), and Ruthenium Red (Sigma Chemical Co.). The commercial product contains approximately 20 % of the dye, the rest being NaCl. The concentrations were calculated accordingly. The mitochondrial fraction of nerve terminal volume was estimated by cutting and weighing profiles of 78 cross-section electromicrographs of two neuromuscular junctions. The areas of the mitochondrial and the nerve terminal cross-sections were measured separately, and the terminal volume calculated knowing the total length of the unmyelinated part of the nerve-terminal arborizations in the endplates from previous light micrographic measurements. RESULTS
The effect of non-8pecific mitochondrial inhibitors on transmitter release Mitochondria are able to take up calcium ions from their environment by a number of processes (Carafoli & Rossi, 1971; Carafoli, Gazzotti, Rossi & Tiozzo, 1971). Two of them, namely the binding to the high as well as low affinity sites, do not need metabolic energy. In addition, there is an energy dependent uptake process for calcium (see Lehninger, 1970). When the process of energy production is inhibited, not only is the calcium uptake ability of the mitochondria greatly reduced, but they may even lose the already sequestrated calcium. The resulting increase in intracellular [Ca] ([Ca]1n) would be expected to raise the level of spontaneous transmitter release. If one examines previous experiments on nerve terminals deprived of their energy supply, the frequency of the spontaneous m.e.p.p.s. was increased in all cases (Kraatz & Trautwein, 1957; Hubbard & L0yning, 1966; Glagoleva, Liberman & Khashayev, 1970; Katz & Edwards, 1973). An illustration of such a phenomenon is given in Fig. 1. Several minutes after the addition to the perfusing fluid, of a dicoumarol (sodium warfarin), which uncouples oxidative phosphorylation, the frequency of the spontaneous m.e.p.p.s. increased more than tenfold. Although the effect of dicoumarol on m.e.p.p. frequency is clear cut, the interpretation of this result is not. The uptake of calcium by mitochondria is not the only energy requiring mechanism in the presynaptic nerve terminal. Among other processes, the maintenance of the resting potential also requires energy. Hence, it is possible that the increase in m.e.p.p. frequency after dicoumarol is due to membrane depolarization of the terminals, which is known to lead to a very substantial increase in
288Q!
E. ALNAES AND R. RAHAMIMOFF
A
B
L Fig. 1. Effect of dicoumarol on m.e.p.p. frequency. A: control. B: after application of 2-5 mm dicoumarol in perfusing solution. Calibration bars: 1 mV, 20 msec.
m.e.p.p. frequency (del Castillo & Katz, 1954c). Depolarization of the terminals cannot be measured at the frog neuromuscular junction. However, indirect evidence suggests that this is not the primary mode of action of dicoumarol. Depolarization induced release depends to a large extent on the presence of calcium ions in the bathing medium. In the absence of external calcium ions, depolarization has only a small effect on m.e.p.p. frequency. If, on the other hand, the increase in spontaneous release is mainly secondary to calcium leakage from the mitochondria, the presence or the absence of extracellular calcium would have no great influence. Fig. 2 shows that dicoumarol increases the rate of the spontaneous events even in the absence of extracellular calcium.
MITOCHONDRIA AND TRANSMITTER RELEASE
289
30 r
20 I0
10 00 00 00
u
4
V)
0
3 .
21_ a
iI*
b
~*00S.00 * @0.0%
I.. 0000 _
*
0
0 -
P9%
*
0.0.
.
I
10
20
|
30 Min
C.|
40
50
60
Fig. 2. Dicoumarol effect on m.e.p.p. frequency in calcium-free Ringer solution. a: change of control solution. b: addition of 250 pI dicoumarol. c: addition of 2-5 mM dicoumarol.
Effect of dicoumarol on evoked quantal release Another way of excluding the possibility that the main effect of dicoumarol on spontaneous m.e.p.p. frequency is due to presynaptic depolarization is to examine its effect on evoked release. It has been shown directly at the squid giant synapse (Katz & Miledi, 1967 b) and indirectly at the vertebrate neuromuscular junction (Hubbard & Willis, 1962), that after depolarization the action potential is less effective in inducing transmitter release. If dicoumarol exerts its effect on m.e.p.p. frequency by depolarization, then it would be expected that the evoked transmitter release would be diminished after dicoumarol. If, on the other hand, the action of dicoumarol results from leakage of calcium from the
290 E. ALNAES AND R. RAHAMIMOFF mitochondria in the nerve terminal, this calcium will add to the calcium entering during the action potential; this would lead to an increase in the total internal [Ca] and potentiation in evoked release. Figs. 3 and 4 show that after moderate dicoumarol poisoning there is an increase in the quantal content, and consequently in the end-plate potential amplitude. The e.p.p. amplitude sometimes can be suprathreshold and lead to contraction (Fig. 3). The increase in quantal content, following dicoumarol is relatively short lived. After about j-1 hr (depending on B
A
C
4' msec Fig. 3. Effect of dicoumarol on evoked transmitter release. A: control series of e.p.p.s in high magnesium low calcium Ringer solution. B: 7 min after addition of 809W dicoumarol to perfusate. C: 20 min after addition. Muscle fibres in the vicinity of the impaled fibre now contract upon nerve stimulation, due to the increase in their e.p.p.s (note contraction artifact in two of the traces).
dicoumarol concentration) there is a failure in e.p.p. formation, presumably as a result of blockage of invasion of the motor nerve terminals by the action potential (cf. Krnjevi6 & Miledi, 1959; Hubbard & L0yning, 1966). Fig. 4 shows graphically the increase in m.e.p.p. frequency and in quantal content following the addition of 80 gM dicoumarol. Dicoumarol is known to be a very potent inhibitor of mitochondrial metabolism. We also tested other mitochondrial inhibitors. The action of other mitochondrial inhibitors on transmitter release Guanidine facilitates myoneural transmission and it has been used clinically to improve depressed neuromuscular transmission (Feng, 1938; Minot, Dodd & Riven, 1938; Otsuka & Endo, 1960; Cherington & Ryan,
MITOCHONDRIA AND TRANSMITTER RELEASE 291 1968; Scaer, Tooker & Cherington, 1969). Guanidine has also been shown to have an inhibitory effect on mitochondria (Pressman, 1963; Chance & Hollunger, 1963). Fig. 5 illustrates that guanidine increases the quantal content of the e.p.p. and the frequency of the spontaneous m.e.p.p. Not only uncoupling agents augment transmitter release. Similar observations were made using mitochondrial inhibitors that act at the electron transport chain level (see Fig. 12 in the Discussion). Thus rotenone (20 /tM) and antimycin (200 /TM) increase the frequency of the spontaneous m.e.p.p., that becomes more pronounced after nerve stimulation (Fig. 6). 15
m
10~~~~~~~~~~~~ 10
.
0*
*
.*.-~~~~5
.5
*
..*.@-.**. .... f 1
0
11o
..
00
5
10
15
20
Fig. 4. Dicoumarol effect on spontaneous and evoked transmitter release. Each point denotes the average quantal content (m) of thirty consecutive nerve stimulations in high magnesium (10 mM) low calcium (0.7 mM) solution. Lower curve shows corresponding changes in the frequency of miniature end-plate potentials (right-hand ordinate). Arrow marks addition of 80 /ZM dicoumarol.
Ruthenium red and release of transmitter All inhibitors of calcium uptake that have been discussed so far work through a rather generalized energy deprivation of mitochondria. They inhibit a number of energy requiring processes in the mitochondria, such as calcium uptake, ATP production and others. To focus more specificially on calcium uptake by mitochondria, we used the inorganic dye Ruthenium red (RuR) which inhibits both passive and active calcium entry in mitochondria (Moore, 1971; Vasington, Gazotti, Tiozzo & Carafoli, 1972). RuR is widely used for histochemical staining, mainly of mucopolysaccharides (see Luft, 1971 a, b). Since RuR penetrates nerve membranes, presumably through their unmyelinated segments (Singer, Krishnan &
292 E. ALNAES AND R. RAHAMIMOFF Fyfe, 1972) we examined its effect on both spontaneous and evoked transmitter release. Two main questions were posed: (1) does RuR, by blocking calcium transport into nerve mitochondria, cause an increase in spontaneous miniature e.p.p. frequency; (2) if the sole effect of RuR is on calcium transport in mitochondria, one would expect an augmentation of evoked release, similar to that produced by non-specific mitochondrial blocking agents. On the other hand, if the calcium transport process across the surface membrane of the nerve (which presumably is responsible for evoked release), shares common properties with the calcium transport process into the mitochondria, RuR will suppress evoked release. B
A
C
ms
0.0 mm-G
20 mM -G
so mm -G
Fig. 5. Effect of guanidine on evoked and spontaneous transmitter release. A: control series in high magnesium (10 mM) low calcium (0.7 mM) Ringer solution. B: after addition of 2 mm guanidine (G). C: after addition of 5 mM guanidine. Note increase in frequency of spontaneous miniature end-plate potentials, as well as in amplitude and quantal content of evoked end-plate potentials.
Effect of RuR on spontaneous release On examining the effect of RuR on transmitter release, a depressing action of RuR on the amplitude (a) of the spontaneous miniature e.p.p.s. was observed. This inhibitory effect increased with the RuR concentration (Fig. 7), and was observed in 11 other preparations. In two experiments done in 40,uM-RuR, any synaptic activity was undistinguishable from
MITOCHONDRIA AND TRANSMITTER RELEASE A
8
C
D
E
F
293
40 msec 2
m
V
Fig. 6. Effect of antimycin on transmitter release. A: control series of stimulations in high magnesium low calcium Ringer solution. B: immediately after addition of 05 mm antimycin to extracellular fluid. C: 1 min later, at 0-5 see'. D: double stimulation at 1 0 sec1 which brings about a very large increase in m.e.p.p. frequency. E: immediately after resuming single stimulation at original low rate (05 sec"). F: 2 min after resuming control stimulation parameters. Quantal content in the control situation was 1 1. After 15 min in antimycin, the quantal content was 1D95.
nerve
E. ALNAES AND R. RAHAMIMOFF the noise. This presumed post-synatpic effect of RuR imposed an upper limit on the useful concentration ranges. Reduced amplitude of the m.e.p.p. can arise from an increase in the membrane conductance of the muscle fibre (Katz & Thesleff, 1957). Such an explanation does not apply to the action of RuR, since in two experiments it was found that the input resistance of the post-synaptic muscle fibre increased upon addition of RuR. 294
0-8 F
06 F
0 i
(MY)
.
04
0*2 .
Ulum
0
*
II
2-5
5-0
7.5
PM
Fig. 7. Post-synaptic effect of Ruthenium red (RuR). Average miniature end-plate potential amplitude a measured at the same neuromuscular junction at three different RuR concentrations.
The effect of RuR (5 pM) on m.e.p.p. frequency was examined in eight experiments in which the initial amplitude of the spontaneous events was large enough so that, even after the depression caused by RuR, the m.e.p.p.s would still be clearly distinguishable from the noise. It was found that on the average there was approximately a doubling in m.e.p.p. frequency after 15-20 min exposure to RuR (range 146-400 % of the control).
MITOCHONDRIA AND TRANSMITTER RELEASE
295
Evoked transmitter release and ruthenium red RuR has a very strong inhibitory effect on e.p.p. amplitude. Addition of 5-10 /LM-RuR to normal Ringer, with 1'8 mm calcium, is usually sufficient to block normal transmission across the neuromuscular synapse and prevent contractions. Analysis of this action shows that the main effect of RuR is on evoked transmitter release; the quantal content is B
A
2 mV
40 msec
Fig. 8. Effect of Ruthenium red (RuR) on evoked and spontaneous transmitter release. A: control series of nerve stimulations in high magnesium low calcium-Ringer solution. B: 10 min after addition of 5 /SM-RuR to extracellular fluid. Observe decrease in quantal content and m.e.p.p. amplitude, as well as increase in m.e.p.p. frequency.
very substantially decreased after RuR (Fig. 8). The effect of RuR in reducing evoked release of transmitter was seen in seventeen preparations under various experimental conditions. A prolonged wash in RuR-free medium only partially restores transmitter liberation. The inhibitory action of RuR on evoked release of transmitter does not arise from inability of the terminals to liberate quanta of acetylcholine
E. ALNAES AND R. RAHAMIMOFF 296 upon the arrival of the nerve impulse. This can be shown by increasing the calcium concentration in the external medium. Fig. 9 illustrates such an experiment. Neuromuscular transmission was blocked by addition of 1OpM-RuR to standard Ringer (1.8 mm calcium) and the resulting subthreshold e.p.p. amplitude was on the average 2 mV. Doubling [Ca] (at the first bar) and quadrupling it (at the second bar) produced a very substantial increase in e.p.p. amplitude and quantal 12
10 8 -~~~~~~~~~:
8 mV 6
4.
2
m
_
0
12 10 8 6 Min Fig. 9. Calcium-induced increase in e.p.p. amplitude (and quantal content) when neuromuscular transmission has been suppressed by 10 /laM Ruthenium red. Extracellular Ca-concentration doubled successively at the two filled bars. Each point represents the mean amplitude of ten responses. See text for further details. 0
2
4
content (the quantal contents at the steady-state level were 21-7, 42-5, and 112 respectively, estimated from the variance; cf. Martin 1955). If the concentration of calcium in the extracellular medium was increased to 14.4 mm (8 times the standard), evoked release rates reached values of about 250 (257.6) in the presence of RuR, which are in the normal range of release (Katz, 1962). However, the e.p.p. amplitudes were still subthreshold, due to the post-synaptic inhibitory action of RuR.
MITOCHONDRIA AND TRANSMITTER RELEASE
297
A
0-4
0
03 0 as
0
0
pm2 02[
0 0
0
0
.
01 _
. * *
0 0 0 0~
*
.
0
U *
0
.
0
0
*0He
*0 C0 'moe
*Uh
U-
40r
*
0
* -
a
I*
I
5
-2
I
__j
I
2
3 pm2
4
5
6
B 0
30 0.
*
a
0
*@-
U *
0
0
10
0~~~~ 0
No .
EU 0
.
0
*
*
0
- I AV
a
I
0
*
A l_ Vi
*"*
0
-
I
'*
m
* -
I |
I __
I
6 5 3 4 PM2 Fig. 1OA. Mitochondrial cross-sectional area-Am (ordinate) plotted against nerve terminal area-An (abscissa) in seventy-eight cross-sections of unmyelinated portion of presynaptic nerve terminals in two different muscles (squares and circles). B: mitochondrial cross-sectional area (ordinate) plotted as percentage of nerve terminal area (abscissa). Same data as in A. 0
1
2
298
E. ALNAES AND R. RAHAMIMOFF
Mitochondrial content of nerve terminal The presence of large numbers of mitochondria in nerve terminals has long been recognized (see Birks, Huxley & Katz, 1960). To obtain quantitative information we used seventy-eight electron micrographs taken from random sections through nerve terminals of two muscles. The micrographs were kindly provided by Mr Kenneth Fischbeck. The terminals were enlarged 63000 x, cut out of the electron microscopic photographs and the paper was weighed. From the 'weight' of 1 /um2, in the photographs, one can convert the 'weights' of the terminal profiles into cross-sectional areas. A similar procedure was repeated for the mitochondria. The results are presented in Fig. 10 in two ways. In Fig. 10A the area of all the mitochondria in a given section (Am) is plotted against area of the nerve terminal cross-section (A.). The number of mitochondria in Am varied between 0 and 8. Fig. 10B is based on the same data as 10A, but shows the fractional occupation by the mitochondria, the ordinate being (Am/An) x 100. There is a considerable variation in the mitochondrial content of nerve terminal sections, the range being between 0 and 38-6 %. On the average the mitochondria occupy 6-59 % of the nerve terminal. DISCUSSION
The basic assumption in this discussion is that the calcium concentration inside the nerve terminal controls transmitter release (see Katz & Miledi, 1967a, b). The exact location of the action of intracellular ionized calcium is still unknown, and for simplicity we have assumed that the free calcium ion concentration near the inside of the presynaptic membrane determines transmitter release. (The qualitative interpretation of the experimental results will be similar if we assume that release of transmitter depends on the surface density of calcium ions on the inside of the presynaptic membrane; which in turn will depend on the free intracellular calcium concentration.) The free intracellular calcium concentration will depend on a balance between several processes: (1) the resting calcium influx and the calcium influx following depolarization (Baker, Hodgkin & Ridgway, 1971) which raises [Ca]in; (2) the opposing calcium efflux (Baker & Blaustein, 1968; Blaustein & Hodgkin, 1969; Baker, Blaustein, Hodgkin & Steinhardt, 1969; DiPolo, 1973; Mullins & Brinley, 1975) which lowers [Ca]in; (3) intracellular calcium buffers such as the mitochondria (Lehninger, 1970); and other calcium binding systems (see Hillman & Llinas, 1974; Oschman, Hall, Peters & Wall, 1974), that are able to bind calcium reversibly. Thus, the regulation of the intracellular calcium
MITOCHONDRIA AND TRANSMITTER RELEASE 299 concentration is a complex process, depending on the contributions of the various components (see schematic representation in Fig. 11). In the present work we tried to examine the role of one of the components in this scheme, namely the mitochondria.
[CO+]
0 0
Fig. 11. Factors which influence the intracellular calcium level which in turn may determine transmitter release. See text for further discussion.
Several lines of evidence suggest that mitochondria can indeed play a part in transmitter release. Mitochondria are abundant in the nerve terminal portion of the nerve; they occupy about 6 % of the volume. This figure is significantly higher than the figure of 1 % found in the squid giant axon (DiPolo, 1973). The average length of the terminals in the data shown in Fig. 10 was approximately 700 gtm (688 + 204; mean + S.D.) and the mean cross-section P5 /Zm2. Hence, the mean volume of the terminal will be approximately 1050 ,nm3. With a value of 6 % mitochondrial mass, one obtains 63 /um3 of mitochondria in the terminal. If the specific gravity of mitochondria is about 1 and the mitochondrial mass contains about 10 % protein, and the calcium binding is 300 x 10-9 mole/mg protein, then the mitochondrial calcium binding capacity is approximately 1P9 x 10-15 mole calcium per terminal. This value exceeds by several orders of magnitude the amount of calcium that enters the nerve terminal due to an action potential; it has to be recognized, though, that the values were taken from different species, under various experimental conditions; nor do we know at present the extent of calcium binding under physiological conditions. However, there is little doubt that calcium binding capacity of the mitochondria well exceeds the influx brought about by an action potential. The response of the nerve terminal to mitochondrial inhibition is rather uniform. Regardless of the point of attack within the mitochondrial metabolism, the terminals increase their level of spontaneous transmitter
E. ALNAES AND 1. RAHAMIMOFF release. Thus, the same result if one inhibits the mitochondria at the electron transport chain level by rotenone, antimycin, cyanide or lack of oxygen (Hubbard & L0yning, 1966; Katz & Edwards, 1973; Alnaes, Rahamimoff, Rotshenker & Shimoni, 1974; Alnaes, Meiri, Rahamimoff & Rahamimoff, 1974) or at the coupling stage (Glagoleva, Liberman & Khashaiev, 1970; Katz & Edwards, 1973; Rahamimoff & Alnaes, 1973), namely an increase in the spontaneous release rate (Fig. 12). 300
Succinate
Glutamate NAD
____
Pyruvate
Calcium uptake
>
A \I / B
CoQ, cyt b -
72
ADP
2
Ascorbate X C D cyt c1-cyt c- cyt a: a3-
2
33
ATP
Fig. 12. Metabolic pathways of mitochondria. Electron transport proceeds along upper horizontal arrows. One (marked (1)) of three ATP generating side branches is drawn schematically. Calcium uptake is thought to occur facultatively to ADP-ATP conversion. Probable sites of action of mitochondrial inhibitors: A: barbiturates, rotenone. B: antimycin. C: cyanide. D: hypoxia. E: uncoupling agents (e.g. dicoumarol, DNP). F: specific calcium uptake antagonists (e.g. Ruthenium red) (after Lehninger, 1970; Carafoli et al. 1971; Carafoli & Rossi, 1971).
It seems simplest to assume that the increase in the spontaneous release of transmitter results from an increase in the intracellular calcium concentration in the terminals. This in turn can arise from a disturbance in the balance between influx and efflux through the presynaptic membrane, uptake by the mitochondria, calcium leakage from mitochondria or other calcium binding intracellular structures. These processes cannot be separately assessed with present methods at the neuromuscular junction. But the fact that an increase in spontaneous release of transmitter can be observed even in the absence of appreciable amounts of calcium in the extracellular fluid suggests that the main source of calcium for this elevated spontaneous release is intracellular. Not only spontaneous but also evoked release of transmitter is augmented by mitochondrial inhibitors. If evoked release is a function of the calcium influx following the action potential and represents the release due to the sum of the existing intracellular calcium plus that newly entered, then after inhibition of mitochondrial calcium uptake, this quantity will be greater, leading to augmentation of evoked release.
MITOCHONDRIA AND TRANSMITTER RELEASE 301 A highly non-linear power relation has been observed between the extracellular calcium concentration and evoked release of transmitter (Jenkinson, 1957; Dodge & Rahamimoff, 1967; Katz & Miledi, 1969; Hubbard, Jones & Landau, 1968). It is not clear at present whether this non-linearity represents the relation between the extracellular calcium concentration and calcium entry, or between the intracellular calcium concentration and release. In the latter case, mitochondrial inhibition and the subsequent inhibition of calcium uptake would be very effective in determining the level of transmitter release following the action potential; thus a metabolic control of synaptic transmission can be envisaged. If the main determining factor in the non-linear relation resides in the entry process, such metabolic control will be less efficient. In any case, a power function, with an exponent greater than 1, presumably exists between [Ca]jin and transmitter release: otherwise, the evoked release will not be potentiated by more than the corresponding fraction of the extra spontaneous release that occurs during the e.p.p. (Fig. 4). Two main presynaptic effects of RuR were observed, an increase in the frequency of the spontaneous miniature e.p.p.s and a decrease in the quantal number. The first observation can be explained on the basis of the known inhibitory effect of RuR on calcium uptake by the mitochondria (Moore, 1971; Vasington et al. 1972). The observed decrease in quantal content can be accounted for, if RuR inhibits the calcium transport into the nerve terminal, similarly to its inhibition of the calcium transport into the mitochondria. The increase in m.e.p.p. frequency produced by the lanthanide praseodymium, and the biphasic action on evoked release, may have a similar origin (cf. Alnaes & Rahamimoff, 1974). It should be stressed that these two calcium transport mechanisms are different in many respects. While the first one apparently operates along the electrochemical gradient (see Katz & Miledi, 1969; Baker, Hodgkin & Ridgway, 1971; Baker, 1972; Baker, Meves & Ridgway, 1971, 1973) between the outside and the inside of the nerve terminal, the second process is an energy requiring transport process. However, it has been shown that a passive binding, presumably to the high affinity sites at the mitochondrial membrane, is a necessary step in the calcium uptake by the mitochondria (Chance, 1965; Scarpa & Azzone, 1968). GomezPuyou, Marietta de Puyou, Becker & Lehninger (1972) have further shown that the effect of RuR on calcium uptake can be accounted for by its action on the glyco-protein molecules, isolated from mitochondria, which have properties similar to the high affinity binding sites. It will be of interest to see whether glycoprotein molecules participate in the calcium entry process into the nerve terminal. After the e.p.p. the probability of release of quanta of transmitter
302 E. ALNAES AND R. RAHAMIMOFF remains at an abnormally high level for several hundreds of milliseconds, and is manifested as the so-called delayed release of spontaneous miniature e.p.p.s (Miledi & Thies, 1971; Rahamimoff & Yaari, 1973) as well as an increase in the number of quanta released by a second nerve impulse (facilitation; del Castillo & Katz, 1954d). It was proposed that this increased probability of transmitter quanta is due to residual calcium (Katz & Miledi, 1965, 1968; Rahamimoff, 1968, 1973). The observation showing that calcium uptake by mitochondria is a fast process starting to operate within tens of milliseconds after exposure (cf. Chance, 1965; Chance, Salkowitz & Kovach, 1972), suggests that mitochondria may take part in the removal of this residual release process. Furthermore, while calcium and strontium are readily taken up by mitochondria, barium is not. When barium activates transmitter release (Miledi, 1966; Alnaes, Meiri, Rahamimoff & Rahamimoff, 1974) delayed release lasts for 8-20 sec, a period much longer than that observed when calcium and strontium activate release. Thus, the residual calcium may be due to at least three processes - delayed entry of calcium (Stinnakre & Tauc, 1973), efflux of calcium through the presynaptic membrane and sequestration by mitochondria. Another aspect of transmitter release in which mitochondria may be involved is the effect of osmotic pressure. It has been shown by Fatt & Katz (1952) and Furshpan (1956) that hyperosmotic media increase spontaneous transmitter release. This finding fits with the observation of Scarpa & Azzone (1968) that the calcium uptake by mitochondria decreases with increase in osmotic pressure in the incubating medium. In developing synapses, delayed release is very high (Fischbach, 1972). This seems to fit with the observation that growing cones and developing terminals have a low mitochondrial content (Tennyson, 1970; Yamada, Spooner & Wessels, 1971; Bunge, 1972), and therefore probably a low calcium-buffering capacity. All these observations suggest that mitochondria play an important part in the regulation of intracellular calcium concentration in the nerve terminal, thereby influencing transmitter liberation and synaptic transmission. Moreover, the abundance of mitochondria in some presynaptic terminals within the central nervous system (cf. Walsh, Houk, Atluri & Mugnaini, 1972), as well as their presence near the vesicles in some sensory cells, such as the chemoreceptors in the carotid body (cf. Al-Lami & Murray, 1968; Biscoe, 1971; Eyzaguirre & Nishi, 1974) and hair cells in the acoustico-lateral line system (Flock, 1965), makes one wonder whether such a regulation of neurosecretion is not of a more generalized occurrence.
MITOCHONDRIA AND TRANSMITTER RELEASE
303 We are indebted to Mr Kenneth Fishbeck for providing the EM photographs and to Dr U. J. McMahan for morphological guidance. We are very grateful to Professor S. W. Kuffler for many valuable discussions throughout the course of the work and during the preparation of the manuscript and for his hospitality. We thank Professor B. Katz for reading the manuscript and for valuable suggestions. This work was supported by N.I.H. and by the Sloan Foundation. REFERENCES
AL-L~.i, F. & Muiuu~r, R. C. (1968). Fine structure of the carotid body of normal and anoxic cats. Anat. Rec. 160, 718. AImA"s, E., MEnui, U., RATHAMIMOFF, H. & RAHAMIoFF, R. (1974). Possible role of mitochondria in transmitter release. J. Phy8iol. 241, 30 P. ALNAE5, E. & RAH moir, R. (1974). Dual action of Praseodynium (Pr+++) on transmitter release at the frog neuromuscular synapse. Nature, Lond. 247, 478479.
ALNAEs, E., RAWANornior, R., ROTSHENKER, S. & SHMOIoa, Y. (1974). Mitochondrial inhibitors and transmitter release at the frog neuromuscular junction. Proc. Israel Physiol. & Pharmacol. Soc. Al 31st meeting. I~rael J. med. Sci. (in the
Press). BAXR, P. F. (1972). Transport and metabolism of calcium ioits in nerve. Prog. Riophy8. molec. Biol. 24, 177-223. BAKER, P. F. & BLAusTEI.N, M. P. (1968). Sodium dependent uptake of calcium by crab nerve. Biochim. biophy8. Acta 150, 167. BAKER, P. F., BL~usTEiN, M. P., HODGKIN, A. L. & STEINHARDT, R. A. (1969). The influence of calcium on sodium efflux in squid axons. J. Phyeiol. 200, 431458.
BmmxR, P. F., HODGKIN, A. L. & RIDGwAY, E. B. (1971). Depolarization and calcium entry in giant squid axons. J. Phyeiol. 218, 709-755. BARER, P. F., MEv:Es, H. & RIDGwAY, E. B. (1971). Phasic entry of calcium in response to depolarization of giant axon of Loligo forbeai. J. Phy8iot. 216, 70P. BAKECR, P. F., MEvEs, H. & RIDGwAY, E. B. (1973). Effects of manganese and other agents on the calcium uptake that follows depolarization of squid axons. J. Phyeiol. 231, 51 1-526. Bmx~s, R. I., HuxL~EY, H. E. & KATz, B. (1960). The fine structure of the neuromuscular junction of the frog. J. Physiol. 150, 134-144. BiscoE, T. J. (1971). Carotid body: structure and function. Phyeiol. Rev. 51, 437-495.
BLAusTErN, M. P. & HODGKI, A. L. (1969). The effect of cyanide on the efflux of calcium from squid axons. J. Phyeiol. 200, 497-527. BUNGEM,M. B. (1972). Fine structure of nerve fibers and growth cones in isolated sympathetic neurons in culture. J. cell Biol. 56, 713-735. CAIRAoLI, E., GAzzoTTI, P., Rossi, C. S. & Tiozzo, R. (1971). Ca++ and mitochondrial membranes: evidence for specific enzymic carriers. Adv. exp. Med. & Biol. 14, 63-85. CAFA~orLi, E. & Rossi, C. 5. (1971). Calcium transport in mitochondria Adv. Cytopharmacol. 1, 209-227. CEhkNcE:, B. (1965). The energy-linked reaction of calcium with mitochondria. J. biol. Chem. 240, 2729. CHAN,-CE, B. & HoLLuNGEHR, G. (1963). Inhibition of electron and energy transfer in mitochondria: IIL The site and the mechanism of guanidine action. J. biol. Chem. 238, 432-438.
304
E. ALNAES AND R. RAHAMIMOFF
CHANCE, B., SAL.OVITZ, I. & KOVACH, A. G. (1972). Kinetics of mitochondrial flavoprotein and pyridine nucleotide in perfused heart. Am. J. Physiol. 223, 207-218. CHERINGTON, M. & RYAN, D. W. (1968). Botulism and guanidine. New Engi. J. Med. 278, 931-933. DEL CASTILLO, J. & KATZ, B. (1954a). The effect of magnesium on the activity of motor nerve endings. J. Phy8iol. 124, 553-559. DEL CASTILLO, J. & KATZ, B. (1954b). Quantal components of the end-plate potential. J. Physiol. 124, 560-573. DEL CASTILLO, J. & KATZ, B. (1954c). Statistical factors involved in neuromuscular facilitation and depression. J. Physiol. 124, 574-585. DEL CASTILLO, J. & KATZ, B. (1954d). Changes in end-plate activity produced by presynaptic polarization. J. Physiol. 124, 586-604. DEL CASTILLO, J. & STARK, L. (1952). The effect of calcium on the motor end plate potentials. J. Physiol. 116, 507-515. DODGE, F. A. & RAHAMIMOFF, R. (1967). Co-operative action of calcium ions in transmitter release at the neuromuscular junction. J. Physiol. 193, 419-432. DIPOLO, R. (1973). Calcium efflux from internally dialysed squid giant axons. J. gen. Physiol. 62, 575-589. ECCLES, R. M., L0YNING, Y. & OSHIMA, T. (1966). Effects of hypoxia on the monosynaptic reflex pathway in the cat spinal cord, J. Neurophysiol. 29, 315-332. EYZAGUIRRE, C. & NIsHI, K. (1974). Further study on mass receptor potential of carotid body chemosensors. J. Neurophysiol. 37, 156-169. FATT, P. & KATZ, B. (1951). An analysis of the end-plate potential recorded with an intracellular electrode. J. Physiol. 115, 320-370. FATT, P. & KATZ, B. (1952). Spontaneous subthreshold activity at motor nerve endings. J. Physiol. 117, 109-128. FENG, T. P. (1936). Studies on the neuromuscular junction. The universal antagonism between calcium and curarizing agencies. Chin. J. Physiol. 10, 513-528. FENG, T. P. (1938). Studies on the neuromuscular junction. X. The effects of guanidine. Chin. J. Physiol. 13, 119-140. FISCHBACH, G. B. (1972). Synapse formation between dissociated nerve and muscle cells in low density cell cultures. Devl Biol. 28, 407-429. FLOCK, A. (1965). Electronmicroscopic and electrophysiological studies on the lateral line canal organ. Acta oto-lar. 199, 90M. FURsHPAN, E. J. (1956). The effects of osmotic pressure changes on the spontaneous activity at motor nerve endings. J. Physiol. 134, 689-697. GLAGOLEVA, I. M., LIBERMAN, Y. A. & KHASHAYEV, Z. (1970). Effect of uncoupling agents of oxidation phosphorylation on the release of acetylcholine from nerve endings. Biofizika 15, 76-83. GomEz-Puyou, A., DE Puyou, MARIETTA BECKER, G. & LEHNINGER, A. (1972). An insoluble Ca++ binding factor from rat liver mitochondria. Biochem. biophys. Res. Commun. 47, 814-819. HILLMAN, D. E. & LLINAS, R. (1974). Calcium containing electron dense structures in the axons of the squid giant synapse. J. cell Biol. 61, 146-155. HUBBARD, J. I., JoNEs, S. F. & LANDAU, E. M. (1968). On the mechanism by which calcium and magnesium affect the release of transmitter by nerve impulses. J. Physiol. 196, 75-86. HUBBARD, J. I. & LoYNING, Y. (1966). The effects of hypoxia on neuromuscular transmission in a mammalian preparation. J. Physiot. 185, 205-223. HUBBARD, J. I. & WILLIS, W. D. (1962). Hyperpolarization of mammalian motor nerve terminals. J. Physiol. 163, 115-137.
MITOCHONDRIA AND TRANSMITTER RELEASE
305
JENKNSON, D. H. (1957). The nature of the antagonism between calcium and magnesium ions at the neuromuscular junction. J. Physiol. 138, 434-444. KATZ, B. (1962). The Croonian Lecture: The transmission of impulses from nerve to muscle and the subeellular unit of synaptic action. Proc. R. Soc. B 155, 455477. KATZ, B. (1969). The release of neural transmitter substances. Sherrington Lecture no. 10, pp. 60. Liverpool: Liverpool University Press. KATZ, N. L. & EDWARDS, C. (1973). Effects of metabolic inhibitors on spontaneous and neurally evoked transmitter release from frog motor nerve terminals. Proc. 26th Meeting Soc. Gen. Physiol. J. gen. Physiol. 61, 259. KATZ, B. & MIIEDI, R. (1965). The effect of calcium on acetylcholine release from motor nerve terminals. Proc. R. Soc. B 161, 496-503. KATZ, B. & MUMEDI. R. (1967a). A study of synaptic transmission in the absence of nerve impulse. J. Physiol. 192, 407-436. KATZ, B. & MILEDI, R. (1967b). The release of acetylcholine from nerve endings by graded electric pulses. Proc. R. Soc. B 167, 23-38. KATZ, B. & MILEDI, R. (1968). The role of calcium in neuromuscular facilitation. J. Physiol. 195, 481-492. KATZ, B. & MLEDI, R. (1969a). The effect of divalent cations on transmission in the squid giant synapse. Publ. Staz. zool. Napoli 37, 303-310. KATZ, B. & MILEDI, R. (1969b). Tetrodotoxin resistant electric activity in presynaptic terminals. J. Phyaiol. 203, 459-487. KATZ, B. & THESLEFF, S. (1957). On the factors which determine the amplitude of the 'miniature end-plate potential.' J. Phyaiol. 137, 267-278. KRAATZ, H. G. & TRAuTwEIN, W. (1957). Die Wirkung von 2,4-Dinitrophenol (DNP) auf die neuromuskulare Erregung subertragung. Arch. exp. Path. Pharmak. 231, 419-439. KRNJEVI6, K. & MILEDI R. (1959). Presynaptic failure of neuromuscular propagation in rats. J. Phyaiol. 149, 1-22. LEHNINGER, A. L. (1970). Mitochondria and calcium ion transport. Biochem. J. 119, 129-138. LUFT, J. H. (1971 a). Ruthenium Red and Violet I. Chemistry, purification, methods for use for electron microscopy and mechanism of action. Anat. Rec. 171, 347368.
LUFT, J. H. (1971 b). Ruthenium Red and Violet II. Fine Structural localization in animal tissues. Anat. Rec. 171, 369-416. MARTIN, A. R. (1955). A further study of the statistical composition of the endplate potential. J. Phyaiol. 130, 114-122. MILEDI, R. (1966). Strontium as a substitute for calcium in the process of transmitter release in the neuromuscular junction. Nature, Lond. 212, 1233-1234. MILEDI, R. (1973). Transmitter release induced by injection of calcium ions into nerve terminals. Proc. R. Soc. B. 183, 421-425. MILEDI, R. & TEIIs, R. E. (1971). Tetanic and post-tetanic rise in frequency of miniature end-plate potentials in low calcium solution. J. Physiol. 212, 245-257. MINOT, A. S., DODD, K. & RIVEN, S. S. (1938). The response of the myasthenic state of guanidine hydrochloride. Science, N.Y. 87, 348-350. MOORE, C. L. (1971). Specific inhibition of mitochondrial Ca++ transport by Ruthenium Red. Biochem. biophy8. Res. Common. 42, 298-305. MuLLINS, L. J. & BRINLEY, F. J. (1975). The sensitivity of calcium efflux from squid axons to changes in membrane potential. J. gen. Physiol. 63, 135-152. OscHMAN, J. L., HALL, T. A., PETERS, P. D. & WALL, B. J. (1974). Association of calcium with membranes of squid giant axon. J. cell Biol. 61, 156-165.
306
E. ALNAES AND R. RAHAMIMOFF
OTSUKA, M. & ENDo, M. (1960). The effect of guanidine on neuromuscular transmission. J. Pharnumc. exp. Ther. 128, 213-282. PREsSMAN, B. C. (1963). The effects of guanidine and alkyl guanidines on the energy transfer reaction of mitochondria. J. biol. Chem. 238, 401-409. RAHAMImoFF, R. (1968). A dual effect of calcium ions on neuromuscular facilitation. J. Physiol. 195, 471-480. RAHAMIMOFF, R. (1973). Modulation of transmitter release at the neuromuscular synapse. In The Neuro8cience8, Third Study Program, ed. Scimrrr, F. 0. & WORDEN, F. G., pp. 943-952. Cambridge, Mass. U.S.A.: MIT Press. RAHA.MOFF, R. & ALNAEs, E. (1973). Inhibitory action of Ruthenium Red on neuromuscular transmission. Proc. natn. Acad. Sci. U.S.A. 70, 3613-3616. RAHAMIMOFF, R. & YAARI, Y. (1973). Delayed release of transmitter at the frog neuromuscular junction. J. Phyaiol. 228, 241-257. SCAER, R. C., TooxER, J. & CHERINGTON, M. (1969). Effect of guanidine on the neuromuscular block of botulism. Neurology, Minneap. 19, 1107-1 1 10. SCARPA, A. & AzzoNE, G. F. (1968). Ion transport in liver mitochondria. IV. The role of surface binding on anaerobic Ca++ translocation. J. biol. Chem. 243, 5132-5138. SINGER, M., KRISHNAN, N. & FYFE, D. A. (1972). Penetration of Ruthenium Red into peripheral nerve fibers. Anat. Rec. 173, 375-390. STINNAKRE, J. & TAuc, L. (1973). Calcium influx in active Aply8ia neurons detected by injected Aequorin. Nature, New Biol. 242, 113-115. TENNYSON, V. M. (1970). The fine structure of the axon and growth cone of the dorsal root neuroblast of the rabbit embryo. J. cell Biol. 44, 62-79. VASINGTON, F. D., GAZZOT, P., Tiozzo, R. & CARAWoLI, E. (1972). The effect of Ruthenium Red of Ca++ transport and respiration in rat liver mitochondria. Biochim. biophy8. Acta 256, 43-54. WALsH, J. V., HOUK. J. C., ATLURI, R. L. & MUGNAINI, E. (1972). Synaptic transmission at single glomeruli in the Turtle cerebellum. Science, N.Y. 178, 881-883. YAMADA, K. M., SPOONER, B. S. & WESSELS, N. K. (1971). Ultrastructure and function of growth cones and axons of cultured nerve cells. J. cell Biol. 49, 614-635.
