- Email: [email protected]
Spontaneous quantal and subquantal transmitter release at the Torpedo nerve-electroplaque junction
Neuroscience Vol. 20, No. 3, pp. 911-921, 1987 Printed in Great Britain
0306-4522/87 $3.00 + 0.00 Pergamon Journals Ltd 0 1987 IBRO
SPONTANEOUS QUANTAL TRANSMITTER RELEASE NERVE-ELECTROPLAQUE
AND SUBQUANTAL AT THE TORPEDO JUNCTION
D. MULLER and Y. DUNANT* Departement
de Pharmacologic,
Centre
Medical
Universitaire,
121 I Geneve
4, Switzerland
Abstract-Focal electrodes were used to record the spontaneous miniature potentials generated on delimited patches of innervated membrane in the Torpedo electric organ. The main population of miniature potentials followed a bell-shaped amplitude distribution. In addition, we observed a second class of spontaneous events that were smaller and whose amplitude distribution was skewed. These sub-
miniatures formed an homogenous population together with the regular miniatures with respect to their time course versus amplitude relationship. They were thus probably generated at the same sites. The proportion of potentials that were subminiature was less than 10% in resting, freshly excised tissue, but it increased markedly: (i) when the tissue was kept for 24-28 h in vitro after excision; (ii) in the period following a brief heat challenge or (iii) stimulation to exhaustion; and (iv) in the presence of dinitrophenol or dinitrofluorobenzene. In all these conditions, we measured the acetylcholine, adenosine 5’-triphosphate and creatine phosphate content of the tissue and found a correlation between the relative number of subminiature potentials and the lack of energy rich molecules. It is concluded that subminiature potentials are present in the electric organ as in neuromuscular junctions. They are probably produced at the same sites as the regular miniature potentials and their relative occurrence seems to increase greatly when the nerve terminals are in a state of energy deficiency.
Miniature potentials are the manifestation of a spontaneous release of subthreshold amounts of transmitter at the neuromuscular junction and other synapses. Miniature potentials are very regular in size and time course and their amplitude corresponds to that of the quanta composing the evoked postsynaptic potential elicited by activation of the afferent nerve.” A second class of spontaneous potentials is also found under certain experimental conditions, i.e. with a favourable signal to noise ratio. The smallest of those subminiature potentials have an amplitude i/7th to ljl5th that of the regular miniature potentials. At rest, they represent only a small percentage of the total number of events but this proportion is greatly increased under certain experimental conditions such as heat challenge, tetanic stimulation, denervation or reinnervation, or treatment with colchicine, botulinum and other toxins, lanthanum ions, etc.‘4~18*29The nature of the changes responsible for this increased proportion of subminiatures is still a matter of conjecture.26 The electric organ of Torpedo is a modified nerve-muscle system which has proved to be an invaluable preparation for the study of cholinergic synaptic transmission, due to the richness of its innervation and the homogeneity of its organization. Recently, using a loose patch clamp technique, we have shown that evoked transmitter release is also
quanta1 in this preparation and exhibits very similar properties to those described in neuromuscular junctions and other cholinergic synapses.‘,** Spontaneous miniature potentials have also been recorded in Torpedo electric organ,6.2o,24 but their quantitative analysis has been limited up to now by two major difficulties: the extreme density of the nerve ending ramifications and the very low input resistance of the electroplaques, which are the postsynaptic cells. These two difficulties were overcome in the present study by using loose patch electrodes to record focally extracellular potentials. It was possible in this way to analyse reproducibly and with a very good signal to noise ratio the spontaneous events generated in small and well-defined patches of innervated electroplaque membrane (20-75 pm’). A short abstract of this work has been published.2’
*Author to whom correspondence
Recording of spontaneous
EXPERIMENTAL
The fish Torpedo marmorata, both males and females, were supplied by the Station de Biologie marine, Arcachon, France. The Torpedo was anaesthetized by tricaine methane sulphonate (0.3 g per I of sea-water) and slices of electric organ were excised and kept in an elasmobranch physiological medium of the following composition: NaCl (280 mM), KC) (7 mM), CaCI, (4.4 mM), MgCI, (I .3 mM), NaHCO, (5 mM), Hepes (20 mM), urea (300 mM), glucose (5.5 mM). This medium was gassed with 95% 0, and 5% CO,; its pH was adjusted to between 7.1 and 7.3. Experiments were carried out at room temperature (18-22”C), unless otherwise stated.
should be addressed.
Abbreviations: ACh, acetylcholine; ATP, 5’-triphosphate; DNP, dinitrophenol; I-fluoro-2,4_dinitrobenzene.
PROCEDURES
A small fragment
adenosine FDNB,
events
of electric organ containing 34 prisms (stacks of electroplaques), was excised and sectioned traversely into a thin slice of tissue. It was then placed with 911
D.
912
MULLER and Y. DUNANT
the innervated faces (ventral faces) uppermost in a small plexiglass chamber coated with Sylgard, and maintained under continuous superfusion with the physiological medium. Recording was done by carefully positioning an extracellular borosilicate glass microelectrode on the superficial electroplaque. The electrodes were produced using a BBCH puller (Mecanex, Geneva). They were broken to a XL.50 pm outer diameter and the tip melted in a microforge. An optimal electrode had a tip inner diameter of 5510pm and a resistance of about 0.3 MR when filled with saline medium. The tip resistance to ground was increased to about 1MQ after the electrode was carefully pressed against the innervated face of an electroplacque and a slight suction applied to the inside of the pipette to improve the signal to noise ratio. The positioning of the electrode was performed under visual control using a binocular microscope. In the case where the innervated face of an electroplaque was on top, spontaneous miniature potentials of regular mean amplitude could be reproducibly recorded at different places on that electroplaque. For each condition tested, the traces from several sites on the same preparation were successively analysed and compared to each other. All traces were stored on a FM tape recorder (HP 3964 A) at 19.05 cm/s, bandwidth DC-5000 Hz. For quantitative analy sis, records were fed into a microprocessor (type SSM, 8080A, San Jose. U.S.A.) and spontaneous events were detected using a trigger equivalent to at least twice the value of the background noise (-20 pV). These spontaneous events were then stored on a floppy disk after 8 bit digi~lization at the equivalent of 30 kHz. They could be checked under visual control and, using a program written in Fortran and previously tested with known artificial signals, the following parameters were calculated: amplitude, time-to-peak, decay time constant and time interval between two events. The events superimposed one upon the other were not considered for these measurements. These data were further stored for statistical analysis and/or display in the form of amplitude histograms, or time course (time-to-peak + decay time constant) versus amplitude relationships. This latter representation was found to be the most sensitive to analyse the homogeneity of the population of spontaneous events recorded by the electrode. Biochemical n?eusuren3ents After the appropriate treatments prisms of electric organ were weighed and their contents extracted in trichloracetic acid during I h at 4°C. The tissues were then homogenized and centrifuged at 8000g during 10min. The supernatant was washed with water-saturated ether and stored at -20°C. Enzyme coupled luminometry was then used to assay the content of samples in acetylcholine,” adenosine 5’-triphosphate (ATP) and creatine phosphate (from Ref. 25 with slight modifications). RESULTS
Amplitude distribution of spontaneous electroplaque potentials Electrical events focally recorded with a loose patch electrode were characterized by a rather broad range of amplitudes, usually from 0.1 to 1 mV. At many places, the spontaneous potentials were seen as a population of homogeneous time course, i.e. the relationship between the time course (rise time plus decay time constant) and the amplitude was monotonic (Fig. I). Their amplitude distribution, however, was not homogenous. One population was distributed in a bell-shaped manner around a mean amplitude of about 0.5-0.8mV, with variations due to
different preparations and recording conditions. Both the times-to-peak and decay time constants of these miniatures increased linearly with amplitude, varying respectively between 0.2-0.4 ms and 0.5-0.8 ms. This population corresponded in size and time course with the elementary constituents, or quanta, composing the neurally evoked electroplaque potential.’ A second population consisted of potentials of small amplitude which were usually less than 10% of the total number of events in freshly excised, resting tissue (Fig. I). Their proportion greatly increased in other circumstances so that the histograms then clearly showed two separate peaks, with the smaller one having emerged from the background noise (Figs 335). The miniature electroplaque potentials with the bell-shaped distribution will be called miniatures, and the smaller events, with the skewed distribution wiil be called subminiatures. In some other preparations, or in other places of the same preparation, the time courses of the spontaneous events were much more heterogeneous. In some cases, two different populations could clearly be distinguished by the different slopes of their time course versus amplitude relationships (Fig. 2). In these cases the amplitude histograms usually showed the presence of two bell-shaped populations. In other cases, more than two populations might have been present and the time course versus amplitude relationships showed then much scattering. These heterogeneous records were interpreted as indicating irregular patch conditions and they were therefore discarded for quantitative analysis in the present work. Even in these cases, however, if the individual populations were considered separately, the distribution of miniatures and subminiatures exhibited similar changes to those observed in records with only one population. The frequencies of spontaneous events showed very broad variations, between a few events per min to a few per s, from one preparation to another, and also from place to place in the same preparation. Furthermore, the occurrence could also vary greatly with time at a given site, with periods of relatively low frequency interrupted by bursts of discharges. We are aware that excision of the tissue, mechanical disturbance with the electrode and other experimental factors could be responsible for these changes, and we only considered as significant those changes in frequency which could be induced repeatedly and had a reproducible time course.
Ageing of the tissue in vitro Pieces of electrogenic tissue can be kept for a long period of time in the elasmobranch saline medium without major functional and structural disturbances. The amount of acetylchohne in the tissue. the transmitter intraterminal compartmentation and the amplitude of the evoked electrical discharge are not significantly altered 24 h or even 48 h after excision from the Torpedo.4,0
Miniature and subminiature potentials in electric organ
913
a)
Events 50-
Q25mY[
030-
20-
y
10-
0
6
I
0-2
0~-4
0.6 '
0.8 '
1 mV
ms
b)
2-
1.60
D
1.2-
D
utttm D
0-8-
°~ 0.4-
o°
0
0 6
0!2
0'.4
0.6 '
0.8 '
'1 mV
Fig. 1. Homogeneous population of spontaneous events in resting, freshly excised tissue. 547 events were recorded at one site using an extracellular loose patch electrode of about 8/~m in inner tip diameter. The mean frequency was 4.2 Hz. The upper graph shows the amplitude distribution and the lower graph the relationship between the amplitude and the time course (time to peak + decay time constant) of the recorded events. The inset illustrates about 50 consecutive events. For convenience, the extracellularly recorded miniature potentials have been represented as upward going signals. However, the bimodal nature of distribution of the spontaneous electroplaque potentials became more apparent as the tissue aged in vitro. Figures 3a and b show that, 24 h after excision, the proportion of subminiatures had considerably increased over that found in fresh tissue (Fig. 1). This feature was observed with four different Torpedoes, the mean proportion of skewed events increased from about
8% at excision to a mean of 26% the day after (see Table 1). In the experiment illustrated in Fig. 3, the mean frequency of spontaneous events was found to vary by about one order of magnitude between different periods of time during the experiment. Analysis of the amplitude distribution of spontaneous events during these low and high frequency periods showed no significant difference in the proportion of
914
D. MULLER and Y. DUNANT
a)
Events 50-
40-
30-
20-
10-
0
6
I
I
I
I
0.2
0.4
0.6
0.8
I
1 mV
ms
b)
2-
1.6
o zl
oo8o
1.2o
0.8
O
a~
0.4-
o
o
oo
6~ 5 ~ o ~
o
o
a
D
0 0
I
I
I
I
0.2
0.4
0.6
0.8
I
1 mV
Fig. 2. Heterogenous population of spontaneous events. In some places, spontaneous events of different time course could be recorded through the same extracellular electrode. The amplitude distribution (a) showed overlapping of two bell-shaped populations and the time course versus amplitude relationship (b) was characterized by two distinct slopes.
Fig. 3. Spontaneous events recorded 24 h after excision. The amplitude distribution of spontaneous events (a) clearly shows a bimodal aspect due to a greater proportion of subminiature potentials than in freshly excised tissue. In this experiment, the frequency of spontaneous events was characterized by a great variability. However, the events recorded during periods of low freqaency [9.1 Hz in (a)] exhibited a similar amplitude distribution to that recorded during periods of high frequency [62 Hz in (c)]. In (b), a fragment of trace recorded during a period of high frequency shows that the subminiatures (arrows) can easily be distinguished from the quantal miniatures. The time course versus amplitude relationships of both miniature and subminiature potentials also presented an homogenous monotonic slope in this experiment (d).
915
Miniature and subminiature potentials in electric organ A
A
e~
-J
--4
0 0
0 0 0 o ~
-0
D
J -c5
-c5
0
)
'0
m a E
•
~q •
~b o
qT o
6
-0 I
I
I
I
I
I
I
ri
~
[
-0
.
I
m
I
I
-c5
i r----
I
I
[-
[
I
~
I
-0
-0
I/1
W
|
~
6
6
6
6
~
~
6
6
6
!
0
Events
a)
2502 mY l .
20M 15-
m
10_
IIXMIhM
IIII
0 i
0.2
d
.4
i
i
0.6
i
0.8
1 mV
25-
~)
20-
15-
10_
rl
0 i
0.2
d
.4
i
i
0.6
0-8
I
1 mV
25"
)
20"
15-
10"
[I ~-
o o:
' 0.2
MMII I
o'. 4
' 0.6
0'8
1' mV
Fig. 4. Effect of a heat challenge on the amplitude distribution of spontaneous events. The amplitude histograms represent the spontaneous events recorded at room temperature (a), 2 rain after a period of 3 rain at 30"C (b), and after 20 rain of recovery (c). 916
a)
Events 50~i.
100 [
40-
]
30-
20-
III
10-
III
Oo
6
I
0.2
00.4
().6
I
0-8
l mV
50-
b)
JL
4030-
20-
10-
0
6
]
0.2
00.4
66
I
0.8
{mY
50-
c)
0-
30-
2010-
0
!
0
I
0.2
0°.4
0.6 '
"o v.o
1 mV
Fig. 5. Effects of FDNB on the amplitude distribution of spontaneous events. FDNB blocked evoked release in 1-2 h and concomitantly, the amplitude distribution of spontaneous events changed until it showed almost exclusively subminiature potentials (c). The histograms in (b) and (c) represent the amplitude distribution of the spontaneous events recorded 20 min and 45 min after treatment with 0.4 mM FDNB. A few quantal miniatures were still present indicating that the function of the postsynaptic membrane was not altered by FDNB at this stage. 917
918
D. MULLERand Y. DUNANT Table 1. Acetylcholine content and energy stores in conditions of increased proportion of subminiatures Condition Control 2448 h after excision
ACh 100 + 3% (14) (I .02 flmol/g) llOk7(9)
ATP
Creatine phosphate
.~.
.~
Subminiatures/ total events (%)
----
100+_2%(14) (I.l4~mol/g)
100 & 3% (14) (IO.4 pmol/g)
8 + 0.9% (9) 5089 events
69 k 5 (9)**
72 k 5 (9)**
26 + 4 (4)** 2 107 events
I-IO min after heat challenge
95 + 8 (7)
79 f 6 (7)**
61 + 5 (7)**
39 * 4 (3)** 765 events
I- IO min after stimulation at IOHz for 3 min
80 rt 6 (9)*
50 _+3 (6)**
25 _t 6 (6)**
49* 10(4)** 767 events
79 + 12(4)*
35 * 9 (4)**
57 & 1I (3)** 421 events
9 * l(4)**
129+ 13(4)*
82 * 4 (2)** 624 events
DNP for 24 h FDNB for 1-2 h
102 + 2 (4) 30 + 6 (4)**
Results (mean f S.E.M.) are expressed as percentages of control values. The numbers in parentheses refer to the number of samples for the biochemical measurements or to the number of different experiments for the proportion of subminiatures. Asterisks indicate results significantly different from control values (*P < olo5, **P < 0.001).
miniatures and subminiatures (Fig. 3a and c). Also, the time course versus amplitude relationships of both populations were very homogeneous and characterized by the same slope (Fig. 3d). These results therefore suggest that both miniatures and subminiatures were generated in the area covered by the electrode and thus probably arose from the same nerve ending ramification. The amplitude distribution during the high frequency period was also interesting since a large number of events was produced during a relatively short time, which reduced the possibility that the recorded parameters were altered by experimental instability. In this condition, the subminiatures appeared as a population with a general skewed distribution, but which was actually composed of multiple peaks. In a small number of cases, multiple peaks were also observed, although not so dearly, in the amplitude dist~bution of the bell-shaped population of miniature potentials (Figs 1, 3 and 4). Heat challenge and tetanic stimulation
At the neuromuscular junction of the frog, a brief period of heating is soon followed by an increase in the frequency of all spontaneous events and an increase in the percentage of subminiatures.15 We repeated this experiment in the electric organ and anaiysed the spontaneous events before, 2 min after and 20 min after a heat challenge at 30°C for 3 min. Changes similar to those reported in frog were observed in three different experiments (Fig. 4). Before heating, the subminiatures were scarce. Two minutes after the challenge, the overall mean frequency rose From about 5 Hz to about 40 Hz and the subminiatures became very numerous. Later, the frequency decreased and the bell-distributed miniatures again represented the major population. Electrical stimulation of the nerves to the electric organ at 10 Hz for 3 min causes a marked “fatigue”
of transmission, which is accompanied by exhaustion of the ATP and creatine phosphate stores of the tissue.’ We recorded spontaneous potentials during the t-10 min which followed such stimulation and found, similarly, that the proportion of subminiatures greatly increased during that period; it reached approximately half of the total number of events (Table I). Poisoned synapses
The above results suggested that the relative increase in subminiatures might in some way be related to the depletion of the energy stores in the tissue. We therefore decided to test the effects of metabolic inhibitors on the preparation. In the presence of dinitrophenol (DNP; 0.2mMf an uncoupler of oxidative phosphorylation, evoked transmitter release was blocked in 3-5 h. At this stage, the acetylcholine (ACh) content was not aitered, but there was a significant reduction in the amount of ATP and a more pronounced decrease in that of creatine phosphate. No significant difference in the frequency of spontaneous potentials was observed with DNP, but the proportion of subminiatures progressively increased during the course of DNP action (Table 1). With a higher concentration of DNP (0.5 mM), the block of transmission occurred more rapidly, the frequency of spontaneous events clearly increased, and a majority of them were also subminiatures. This increase in frequency was independent of the presence of Ca2+ in the bathing medium. Fluorodinitrobenzene (FDNB) is a classical inhibitor of creatine phosphokinase, an enzyme required for the formation of ATP from creatine phosphate and ADP. At a concentration of 0.2 mM, FDNB blocked transmission within 2-3 h and, during the same time, creatine phosphokinase was inhibited by more than 90% (unpublished obser-
Miniature and subminiature potentials in electric organ
919
release. Several observations support the latter explanation. First, the time course of subminiatures was shorter than that of miniatures and for both populations the time course versus amplitude reIayionships followed the same monotonic slope. If they were due to the release of normal sized quanta from the same ele~troplaque but at a distance of the electrode or from eiectrocytes situated underneath, their time course would have been expected to be longer and their time course versus amplitude relationship would have been different. This was actually observed in the few circumstances when two popuIations were clearly present (see Fig. 2). The same change in time course would also have occurred if the subminiatures were due to the release of normal sized quanta at sites distant from the postsynapti~ receptors, for example from nerve endings at sites opposite to the synaptic cleft.” Experiments at the neuromuscular junction where several electrodes were used to explore the releasing sites along the nerve terminal also led to the conclusion that miniatures and subminiatures are produced at the same place. 26~.10 Secondly, in the experiment where the amplitude distributions of spontaneous events during periods of low and high frequency occurrence were compared (Fig. 3), no change in the ratio of miniatures to subminiatures was observed. This is consistent with the view that both miniatures and subminiatures were generated by the same nerve ending ramification. Finally, the experiments where the normal class of miniatures vanished, while the subDISCUSSION miniatures persisted, are difficult to explain if the subminiatures were generated by the release of norThe electric organ in Torpedo is embryologically homologous to neuromuscular systems, but it differs mal sized quanta far from the electrode. From amplitude histograms showing multiple from them in its cytological organization. In the peaks in both the miniature and subminiature popuelectric organ, the nerve endings form an extremely lations, it has been proposed that both classes are ramified network and their presynaptic membrane built up of an integral number of a common small does not show the “active zones” that characterize subunit, the amplitude of which corresponds to the the presumed sites of release in motor nerve termismallest peak of the subminiature population.‘5,‘7,29 nals.2 Also the synaptic vesicles in the Torpedo are While this view has been ~halleng~d,~’ careful statistitwice as large as in the neuromuscular junction. cal analyses has supported the hypothesis of subDespite these structural differences, the electrophysiological aspects of transmitter release in the two units.‘s~‘9The subminiature populations in the present work often displayed multiple peaks (see Fig. 3 and systems have been found to be quite similar. The 5). In some cases, these multiple peaks were also occurrence of spontaneous miniature potentials, the quanta1 composition of the evoked response, and the apparent in the population of bell-distributed miniacalculated number of acetylcholine molecules in a tures (see Figs 1, 3 and 4). However, we did not quantum are the same in electric organ and at the attempt to look for them quantitatively and systemendplate. The present work adds a new similarity: atically, since good stability of the recording condithe demonstration in the Torpedo of a class of small tions is difficult to obtain for a long time with loose minialure potentials resembling in all aspects those patch electrodes. A slight decrease in the negative described in amphibian, mammalian (including man) pressure was able to change the recording conditions. and invertebrate neuromuscular junctions.‘“‘9*2’J7..29 Also the number of recorded events, which is critical The finding was not unexpected, however, since for observing multiple peaks, was possibly not subminiatures have been observed at the sufIicient in all cases. nerve-electroplaque junction of the skate.” Manoeuvres which are known to increase the An important question is whether the subminiature proportion of subminiatures at the neuromuscular potentials are produced by the release of normal sized synapses were also effective in the electric organ. quanta at sites distant from the recording electrode or Subminiatures were abundant during the period folwhether they actually reflect a different type of ACh lowing a heat challenge or a tetanic stimulation, vations). The ATP and ACh contents were greatly decreased by FDNB, but not the amount of creatine pbosphate, which was even increased (Table 1). This is as would be expected since creatine phosphokinase was inactivated. During incubation in the presence of FDNB, the frequency of spontaneous potentials increased by a factor of 10 or 20 and their size distribution changed (Fig. 5). At the time when transmission failed, subminiatures represented 80-90% of the total number of events, and only a few miniatures were still present. These miniatures were of the usual amplitude and time course, which indicated that FDNB did not significantly alter postsynaptic function at this stage {Fig. 5~). The increase in frequency of spontaneous events provoked by FDNB was also observed in the absence of calcium in the bathing medium [solution with I mM ethyleneglycolbis (aminoethylether)tetraacetate and no added calcium]. Table 1 summarizes the results and indicates that the ATP and creatine phosphate contents of the tissue were altered not only with these metabolic poisons, but also in all conditions where we had observed a greater proportion of subminiature potentials, i.e. 24-28 h after excision of the tissue, after a heat challenge or stimulation to exhaustion. These results suggest therefore that the metabolic state of the nerve endings might be important in determining the proportion of subm~niatures among the population of spontaneous events.
920
D. MULLEK and Y. DUNANT
during deterioration of nerve terminals by denervation or by keeping the tissue in Gtro (which also implies a denervation) and under the action of various drugs and toxins such as botulinurn toxin.5.23To test whether alterations of the subminiature to miniature ratio might be linked to changes in either the ACh content or the metabolic state of the tissue, we also analysed the effects of metabolic inhibitors, DNP and FDNB, and determined in all these conditions the ACh, ATP and creatine phosphate contents of the tissue. The results (Table 1) suggest that there was no relationship between the ACh content and the relative number of subminiatures. On the other hand, a better correlation was found with the amount of ATP and creatine phosphate in the tissue. The proportion of subminiatures, in ail conditions tested, seemed to increase proportionately with a reduction in the level of energy rich compounds. This was also true in presence of FDNB, where creatine kinase was inactivated and creatine phosphate could not be used to form ATP, although its concentration in the tissue remains high. Futhermore, in the presence of botulinum toxin, which selectively blocks choiinergic transmission and the occurrence of miniature potentials but not of subminiatures, the contents of ATP and creatine phosphate, but not of ACh. has also recently been found to be reduced.’ It should be kept in mind, however. that the ATP and creatine phosphate measurements were obtained from prisms of electric organ, which include electrocytes and glial ceils in addition to the nerve terminals. Nevertheless, these measurements should at least partly reflect presynaptic changes, since previous work has shown that, during nerve stimulation, about 25% of the energy rich compounds (ATP and creatine phos-
phate) of the tissue are consumed by the nerves endings.’ A number of different hypotheses have been proposed to account for the occurrence of subminiature potentials at the neuromuscular junction (for example: exocytosis of one synaptic vesicle for one subminiature and of several vesicles for one classical miniature, or a partly filled vesicle for a subminiature. or also mechanisms other than vesicle exocytosis, see Ref. 26). In the Torpedo electric organ, biochemical and morphologi~i experiments have suggested that the releasing mechanism might consist of a protein structure inserted in the presynaptic membrane, uses preferentially cytopiastic acetylwhich choiine.“,h.K.Y.i? We propose that this structure might be composed of subunits, which in case of metabolic depletion are activated individually rather than synchronously, so explaining the greater proportion of subminiatures observed in those cases.3 This is, of course, only one possibility and much work will still be needed to determine whether the correlation observed in the present work between the metabolic state of the nerve endings and the proportion of subminiatures results from direct or indirect interactions. In this respect, it will doubtless be fruitful to investigate the biochemical and structural counterparts of these s~lbminiatures in the torpedo electric organ.
.4ckno~I~~~~~zents-- We
thank F. Lo&n for skilful technical assistance, N. Co&t for typing the text and Dr J. Coles for useful suggestions and criticism. This work was supported by the Fonds National Suisse pour la Recherche scientifique (Grant No. X583.0.84) and the Sandoz Foundation.
REFERENCES 1. Chmouliovsky M., Dunant Y. and Hojvat S. (1974)Pre- and postsynaptic utilization of ATP and creatine phosphate at the nerve~le~troplaque junction. J. ~euroche~. 22, 73-75. 2. Couteaux R. and P&cot-Dechavassine M. (1970) V&icules synaptiques et poches au niveau des “zones actives” de la jonction neuromusculaire. C. R. Acad. Sri. Paris 271, 2346-2349. - Dunant Y. (1986) On the mechanism of acetylcholine release. Prog. Neurobiol. 26, 55-92. 4. Dunant Y.. Eder L. and Servetiadis-Hirt L. (1980) Acetylcholine release evoked by single or a few nerve impulses in the electric organ of Torpedo. J. Ph.ysioi., Load. 298, 185-203. 5. Dunant Y., Esquerda J. E., Lo&n F., Marsal J. and Muller D. (In press) Botulinurn toxin blocks quanta1 acetylcholine release and inhibits creatine kinase in the Torpedo electric organ. f. Physiol. 6. Dunant Y., Gautron J., IsraG M., Lesbats B. and Manaranche R. (1972) Les compartiments d’acitylcholine de I’organe blectrique de la Torpille et leurs modifications par la stimulation. J. Neurochem. 19, 1987-2002. 7. Dunant Y. and Muller D. (1986) Quanta1 release of acetylcholine evoked by focal depolarisation at the Torpedo nerve~le~troplaque junction. J. Physiol., Lomd. 379, 461478. 8. Dunant Y., Muller D., ParduEz A.. Jones G. J. and Garcia-Segura L. M. (1984) Augmentation t&s br&ve du nombre de aarticules dans la membrane .prtsynaptiaue . . _ -pendant la transmission d’un influx nerveux. C. R. Acad. Sci. Paris 299, 543-552. 9. Garcia-Segura L. M., Muller D. and Dunant Y. (1986) Increase in the number of presynaptic large intramembrane particles during synaptic transmission at the Torpedo nerve~lectroplaque junction. ~euroscienee 19, 63-79. 10. Gross C. E. and Kriebel M. E. (1983) Skate electric organs generate two classes of spontaneous miniature electrocyte junction potentials. Ahs. &r. Neurosci. 9, 882. Il. Israii M. and Lesbats B. (1981) Continuous determination by a chemiluminescent method of acetylcholine release and compartmentation in Torpen’rielectric organ synaptosomes. J. ;~e~~ocbe~?, 37, 1475-1483. 12. IsraEl M., Manaranche R., Morel N., Dedieu J. C., Gu~ik-Krzyw~cki T. and Lesbats B. (1981) Redistribution of intramembrane particles related to acetylcholine release by cholinergic synaptosomes. J. Ultrmir. Rw. 75, 162. 178. 13. Katz B. (1969) The Release qj’ Neural Transmitter Substances. University Press, Liverpool. 14‘ Kriebel M. E. and Florey E. (1983) Effect of lanthanum ions on the amplitude distribution of miniature endplate potentials and on synaptic vesicles in frog neuromuscular junctions. Neurcl.vcience9, 535-547.
Miniature
and subminiature
potentials
in electric
organ
921
15. Kriebel M. E. and Gross C. E. (1974) Multimodal distribution of frog miniature endplate potentials in adult, denervated and tadpole leg muscle. J. gen. Physiol. 64, 85-103. 16. Kriebel M. E., Hanna R. B. and Pappas G. D. (1980) Spontaneous potentials and fine structure of identified frog denervated neuromuscular junctions. Neuroscience 5, 97-108. 17. Kriebel M. E., Llados F. and Matteson D. R. (1976) Spontaneous subminiature endplate potentials in mouse diaphragm muscle: evidence for synchronous release. J. Physiol., Lond. 262, 553-581. 18. Kriebel M. E., Llados F. and Matteson D. R. (1982) Histograms of the unitary evoked potential of the mouse diaphragm show multiple peaks. J. Physiol., Lond. 322, 21 I-222. 19. Matteson D. R., Kriebel M. E. and Llados F. (1981) A statistical model indicates that miniature end-plate potentials and unitary evoked end-plate potentials are composed of subunits. /. theor. Rio/. 90, 337-363. 20. Miledi R., Mohnoff P. and Potter L. T. (1971) Isolation of the cholinergic receptor protein of Torpedo electric tissue. Nature, Land. 229, 554-557. 21. Miller D. C., Weinstock M. M. and Magleby K. L. (1978) Is the quantum of transmitter release composed of subunits? Nature, Lond. 274, 388-390. 22. Muller D. (1986) Potentiation by 4-aminopyridine of quanta1 acetylcholine release at the Torpedo nerveeelectroplacque junction. J. Physiol., Lond. 379, 461478. 23. Muller D. and Dunant Y. (1985) Subminiature electroplaque potentials are present in Torpedo electric organ. Experientia 41, 824. 24. Soria B. (1983) Properties of miniature post-synaptic currents at the Torpedo rnarrnorafa nerveelectroplaque junction. Quarf. J. exp. Physiol. 68, 189-202. 25. Strehler B. L. (1974) Adenosine-5’-triphosphate and creatine phosphate. determination with luciferase. In Methods in Enzymatic Analysis, Vol. 4, pp. 2112-2115. Academic Press, New York. 26. Tremblay J. P., Laurie R. E. and Colonnier M. (1983) Is the MEPP due to the release of one vesicle or to similtaneous release of several vesicles at one active zone? Brain Res. Rev. 6, 299-314. 27. Vautrin J. and Mambrini J. (1981) Caracteristiques du potentiel unitaire de plaque motrice de la grenouille. J. Physial., Paris 77, 999-1010. 28. Walther C., Hodgkiss J. P. and Hensler K. (1982) Sub-miniature junction potentials in excitatory neuromuscular synapses of the locust. Neurosci. Lett. 34, 27-32. 29. Wernig A. and Stirner H. (1977) Q uantum amplitude distributions point to functional unity of the synaptic “active zone”. Nature, Lond. 268, 82&822. 30. Wernig A. and Motelica-Heino 1. (1978) On the presynaptic nature of the quanta1 subunits. Neurosci. Let/. 8, 231k234.
(Accepfed 5 August 1986)
0306-4522/87 $3.00 + 0.00 Pergamon Journals Ltd 0 1987 IBRO
SPONTANEOUS QUANTAL TRANSMITTER RELEASE NERVE-ELECTROPLAQUE
AND SUBQUANTAL AT THE TORPEDO JUNCTION
D. MULLER and Y. DUNANT* Departement
de Pharmacologic,
Centre
Medical
Universitaire,
121 I Geneve
4, Switzerland
Abstract-Focal electrodes were used to record the spontaneous miniature potentials generated on delimited patches of innervated membrane in the Torpedo electric organ. The main population of miniature potentials followed a bell-shaped amplitude distribution. In addition, we observed a second class of spontaneous events that were smaller and whose amplitude distribution was skewed. These sub-
miniatures formed an homogenous population together with the regular miniatures with respect to their time course versus amplitude relationship. They were thus probably generated at the same sites. The proportion of potentials that were subminiature was less than 10% in resting, freshly excised tissue, but it increased markedly: (i) when the tissue was kept for 24-28 h in vitro after excision; (ii) in the period following a brief heat challenge or (iii) stimulation to exhaustion; and (iv) in the presence of dinitrophenol or dinitrofluorobenzene. In all these conditions, we measured the acetylcholine, adenosine 5’-triphosphate and creatine phosphate content of the tissue and found a correlation between the relative number of subminiature potentials and the lack of energy rich molecules. It is concluded that subminiature potentials are present in the electric organ as in neuromuscular junctions. They are probably produced at the same sites as the regular miniature potentials and their relative occurrence seems to increase greatly when the nerve terminals are in a state of energy deficiency.
Miniature potentials are the manifestation of a spontaneous release of subthreshold amounts of transmitter at the neuromuscular junction and other synapses. Miniature potentials are very regular in size and time course and their amplitude corresponds to that of the quanta composing the evoked postsynaptic potential elicited by activation of the afferent nerve.” A second class of spontaneous potentials is also found under certain experimental conditions, i.e. with a favourable signal to noise ratio. The smallest of those subminiature potentials have an amplitude i/7th to ljl5th that of the regular miniature potentials. At rest, they represent only a small percentage of the total number of events but this proportion is greatly increased under certain experimental conditions such as heat challenge, tetanic stimulation, denervation or reinnervation, or treatment with colchicine, botulinum and other toxins, lanthanum ions, etc.‘4~18*29The nature of the changes responsible for this increased proportion of subminiatures is still a matter of conjecture.26 The electric organ of Torpedo is a modified nerve-muscle system which has proved to be an invaluable preparation for the study of cholinergic synaptic transmission, due to the richness of its innervation and the homogeneity of its organization. Recently, using a loose patch clamp technique, we have shown that evoked transmitter release is also
quanta1 in this preparation and exhibits very similar properties to those described in neuromuscular junctions and other cholinergic synapses.‘,** Spontaneous miniature potentials have also been recorded in Torpedo electric organ,6.2o,24 but their quantitative analysis has been limited up to now by two major difficulties: the extreme density of the nerve ending ramifications and the very low input resistance of the electroplaques, which are the postsynaptic cells. These two difficulties were overcome in the present study by using loose patch electrodes to record focally extracellular potentials. It was possible in this way to analyse reproducibly and with a very good signal to noise ratio the spontaneous events generated in small and well-defined patches of innervated electroplaque membrane (20-75 pm’). A short abstract of this work has been published.2’
*Author to whom correspondence
Recording of spontaneous
EXPERIMENTAL
The fish Torpedo marmorata, both males and females, were supplied by the Station de Biologie marine, Arcachon, France. The Torpedo was anaesthetized by tricaine methane sulphonate (0.3 g per I of sea-water) and slices of electric organ were excised and kept in an elasmobranch physiological medium of the following composition: NaCl (280 mM), KC) (7 mM), CaCI, (4.4 mM), MgCI, (I .3 mM), NaHCO, (5 mM), Hepes (20 mM), urea (300 mM), glucose (5.5 mM). This medium was gassed with 95% 0, and 5% CO,; its pH was adjusted to between 7.1 and 7.3. Experiments were carried out at room temperature (18-22”C), unless otherwise stated.
should be addressed.
Abbreviations: ACh, acetylcholine; ATP, 5’-triphosphate; DNP, dinitrophenol; I-fluoro-2,4_dinitrobenzene.
PROCEDURES
A small fragment
adenosine FDNB,
events
of electric organ containing 34 prisms (stacks of electroplaques), was excised and sectioned traversely into a thin slice of tissue. It was then placed with 911
D.
912
MULLER and Y. DUNANT
the innervated faces (ventral faces) uppermost in a small plexiglass chamber coated with Sylgard, and maintained under continuous superfusion with the physiological medium. Recording was done by carefully positioning an extracellular borosilicate glass microelectrode on the superficial electroplaque. The electrodes were produced using a BBCH puller (Mecanex, Geneva). They were broken to a XL.50 pm outer diameter and the tip melted in a microforge. An optimal electrode had a tip inner diameter of 5510pm and a resistance of about 0.3 MR when filled with saline medium. The tip resistance to ground was increased to about 1MQ after the electrode was carefully pressed against the innervated face of an electroplacque and a slight suction applied to the inside of the pipette to improve the signal to noise ratio. The positioning of the electrode was performed under visual control using a binocular microscope. In the case where the innervated face of an electroplaque was on top, spontaneous miniature potentials of regular mean amplitude could be reproducibly recorded at different places on that electroplaque. For each condition tested, the traces from several sites on the same preparation were successively analysed and compared to each other. All traces were stored on a FM tape recorder (HP 3964 A) at 19.05 cm/s, bandwidth DC-5000 Hz. For quantitative analy sis, records were fed into a microprocessor (type SSM, 8080A, San Jose. U.S.A.) and spontaneous events were detected using a trigger equivalent to at least twice the value of the background noise (-20 pV). These spontaneous events were then stored on a floppy disk after 8 bit digi~lization at the equivalent of 30 kHz. They could be checked under visual control and, using a program written in Fortran and previously tested with known artificial signals, the following parameters were calculated: amplitude, time-to-peak, decay time constant and time interval between two events. The events superimposed one upon the other were not considered for these measurements. These data were further stored for statistical analysis and/or display in the form of amplitude histograms, or time course (time-to-peak + decay time constant) versus amplitude relationships. This latter representation was found to be the most sensitive to analyse the homogeneity of the population of spontaneous events recorded by the electrode. Biochemical n?eusuren3ents After the appropriate treatments prisms of electric organ were weighed and their contents extracted in trichloracetic acid during I h at 4°C. The tissues were then homogenized and centrifuged at 8000g during 10min. The supernatant was washed with water-saturated ether and stored at -20°C. Enzyme coupled luminometry was then used to assay the content of samples in acetylcholine,” adenosine 5’-triphosphate (ATP) and creatine phosphate (from Ref. 25 with slight modifications). RESULTS
Amplitude distribution of spontaneous electroplaque potentials Electrical events focally recorded with a loose patch electrode were characterized by a rather broad range of amplitudes, usually from 0.1 to 1 mV. At many places, the spontaneous potentials were seen as a population of homogeneous time course, i.e. the relationship between the time course (rise time plus decay time constant) and the amplitude was monotonic (Fig. I). Their amplitude distribution, however, was not homogenous. One population was distributed in a bell-shaped manner around a mean amplitude of about 0.5-0.8mV, with variations due to
different preparations and recording conditions. Both the times-to-peak and decay time constants of these miniatures increased linearly with amplitude, varying respectively between 0.2-0.4 ms and 0.5-0.8 ms. This population corresponded in size and time course with the elementary constituents, or quanta, composing the neurally evoked electroplaque potential.’ A second population consisted of potentials of small amplitude which were usually less than 10% of the total number of events in freshly excised, resting tissue (Fig. I). Their proportion greatly increased in other circumstances so that the histograms then clearly showed two separate peaks, with the smaller one having emerged from the background noise (Figs 335). The miniature electroplaque potentials with the bell-shaped distribution will be called miniatures, and the smaller events, with the skewed distribution wiil be called subminiatures. In some other preparations, or in other places of the same preparation, the time courses of the spontaneous events were much more heterogeneous. In some cases, two different populations could clearly be distinguished by the different slopes of their time course versus amplitude relationships (Fig. 2). In these cases the amplitude histograms usually showed the presence of two bell-shaped populations. In other cases, more than two populations might have been present and the time course versus amplitude relationships showed then much scattering. These heterogeneous records were interpreted as indicating irregular patch conditions and they were therefore discarded for quantitative analysis in the present work. Even in these cases, however, if the individual populations were considered separately, the distribution of miniatures and subminiatures exhibited similar changes to those observed in records with only one population. The frequencies of spontaneous events showed very broad variations, between a few events per min to a few per s, from one preparation to another, and also from place to place in the same preparation. Furthermore, the occurrence could also vary greatly with time at a given site, with periods of relatively low frequency interrupted by bursts of discharges. We are aware that excision of the tissue, mechanical disturbance with the electrode and other experimental factors could be responsible for these changes, and we only considered as significant those changes in frequency which could be induced repeatedly and had a reproducible time course.
Ageing of the tissue in vitro Pieces of electrogenic tissue can be kept for a long period of time in the elasmobranch saline medium without major functional and structural disturbances. The amount of acetylchohne in the tissue. the transmitter intraterminal compartmentation and the amplitude of the evoked electrical discharge are not significantly altered 24 h or even 48 h after excision from the Torpedo.4,0
Miniature and subminiature potentials in electric organ
913
a)
Events 50-
Q25mY[
030-
20-
y
10-
0
6
I
0-2
0~-4
0.6 '
0.8 '
1 mV
ms
b)
2-
1.60
D
1.2-
D
utttm D
0-8-
°~ 0.4-
o°
0
0 6
0!2
0'.4
0.6 '
0.8 '
'1 mV
Fig. 1. Homogeneous population of spontaneous events in resting, freshly excised tissue. 547 events were recorded at one site using an extracellular loose patch electrode of about 8/~m in inner tip diameter. The mean frequency was 4.2 Hz. The upper graph shows the amplitude distribution and the lower graph the relationship between the amplitude and the time course (time to peak + decay time constant) of the recorded events. The inset illustrates about 50 consecutive events. For convenience, the extracellularly recorded miniature potentials have been represented as upward going signals. However, the bimodal nature of distribution of the spontaneous electroplaque potentials became more apparent as the tissue aged in vitro. Figures 3a and b show that, 24 h after excision, the proportion of subminiatures had considerably increased over that found in fresh tissue (Fig. 1). This feature was observed with four different Torpedoes, the mean proportion of skewed events increased from about
8% at excision to a mean of 26% the day after (see Table 1). In the experiment illustrated in Fig. 3, the mean frequency of spontaneous events was found to vary by about one order of magnitude between different periods of time during the experiment. Analysis of the amplitude distribution of spontaneous events during these low and high frequency periods showed no significant difference in the proportion of
914
D. MULLER and Y. DUNANT
a)
Events 50-
40-
30-
20-
10-
0
6
I
I
I
I
0.2
0.4
0.6
0.8
I
1 mV
ms
b)
2-
1.6
o zl
oo8o
1.2o
0.8
O
a~
0.4-
o
o
oo
6~ 5 ~ o ~
o
o
a
D
0 0
I
I
I
I
0.2
0.4
0.6
0.8
I
1 mV
Fig. 2. Heterogenous population of spontaneous events. In some places, spontaneous events of different time course could be recorded through the same extracellular electrode. The amplitude distribution (a) showed overlapping of two bell-shaped populations and the time course versus amplitude relationship (b) was characterized by two distinct slopes.
Fig. 3. Spontaneous events recorded 24 h after excision. The amplitude distribution of spontaneous events (a) clearly shows a bimodal aspect due to a greater proportion of subminiature potentials than in freshly excised tissue. In this experiment, the frequency of spontaneous events was characterized by a great variability. However, the events recorded during periods of low freqaency [9.1 Hz in (a)] exhibited a similar amplitude distribution to that recorded during periods of high frequency [62 Hz in (c)]. In (b), a fragment of trace recorded during a period of high frequency shows that the subminiatures (arrows) can easily be distinguished from the quantal miniatures. The time course versus amplitude relationships of both miniature and subminiature potentials also presented an homogenous monotonic slope in this experiment (d).
915
Miniature and subminiature potentials in electric organ A
A
e~
-J
--4
0 0
0 0 0 o ~
-0
D
J -c5
-c5
0
)
'0
m a E
•
~q •
~b o
qT o
6
-0 I
I
I
I
I
I
I
ri
~
[
-0
.
I
m
I
I
-c5
i r----
I
I
[-
[
I
~
I
-0
-0
I/1
W
|
~
6
6
6
6
~
~
6
6
6
!
0
Events
a)
2502 mY l .
20M 15-
m
10_
IIXMIhM
IIII
0 i
0.2
d
.4
i
i
0.6
i
0.8
1 mV
25-
~)
20-
15-
10_
rl
0 i
0.2
d
.4
i
i
0.6
0-8
I
1 mV
25"
)
20"
15-
10"
[I ~-
o o:
' 0.2
MMII I
o'. 4
' 0.6
0'8
1' mV
Fig. 4. Effect of a heat challenge on the amplitude distribution of spontaneous events. The amplitude histograms represent the spontaneous events recorded at room temperature (a), 2 rain after a period of 3 rain at 30"C (b), and after 20 rain of recovery (c). 916
a)
Events 50~i.
100 [
40-
]
30-
20-
III
10-
III
Oo
6
I
0.2
00.4
().6
I
0-8
l mV
50-
b)
JL
4030-
20-
10-
0
6
]
0.2
00.4
66
I
0.8
{mY
50-
c)
0-
30-
2010-
0
!
0
I
0.2
0°.4
0.6 '
"o v.o
1 mV
Fig. 5. Effects of FDNB on the amplitude distribution of spontaneous events. FDNB blocked evoked release in 1-2 h and concomitantly, the amplitude distribution of spontaneous events changed until it showed almost exclusively subminiature potentials (c). The histograms in (b) and (c) represent the amplitude distribution of the spontaneous events recorded 20 min and 45 min after treatment with 0.4 mM FDNB. A few quantal miniatures were still present indicating that the function of the postsynaptic membrane was not altered by FDNB at this stage. 917
918
D. MULLERand Y. DUNANT Table 1. Acetylcholine content and energy stores in conditions of increased proportion of subminiatures Condition Control 2448 h after excision
ACh 100 + 3% (14) (I .02 flmol/g) llOk7(9)
ATP
Creatine phosphate
.~.
.~
Subminiatures/ total events (%)
----
100+_2%(14) (I.l4~mol/g)
100 & 3% (14) (IO.4 pmol/g)
8 + 0.9% (9) 5089 events
69 k 5 (9)**
72 k 5 (9)**
26 + 4 (4)** 2 107 events
I-IO min after heat challenge
95 + 8 (7)
79 f 6 (7)**
61 + 5 (7)**
39 * 4 (3)** 765 events
I- IO min after stimulation at IOHz for 3 min
80 rt 6 (9)*
50 _+3 (6)**
25 _t 6 (6)**
49* 10(4)** 767 events
79 + 12(4)*
35 * 9 (4)**
57 & 1I (3)** 421 events
9 * l(4)**
129+ 13(4)*
82 * 4 (2)** 624 events
DNP for 24 h FDNB for 1-2 h
102 + 2 (4) 30 + 6 (4)**
Results (mean f S.E.M.) are expressed as percentages of control values. The numbers in parentheses refer to the number of samples for the biochemical measurements or to the number of different experiments for the proportion of subminiatures. Asterisks indicate results significantly different from control values (*P < olo5, **P < 0.001).
miniatures and subminiatures (Fig. 3a and c). Also, the time course versus amplitude relationships of both populations were very homogeneous and characterized by the same slope (Fig. 3d). These results therefore suggest that both miniatures and subminiatures were generated in the area covered by the electrode and thus probably arose from the same nerve ending ramification. The amplitude distribution during the high frequency period was also interesting since a large number of events was produced during a relatively short time, which reduced the possibility that the recorded parameters were altered by experimental instability. In this condition, the subminiatures appeared as a population with a general skewed distribution, but which was actually composed of multiple peaks. In a small number of cases, multiple peaks were also observed, although not so dearly, in the amplitude dist~bution of the bell-shaped population of miniature potentials (Figs 1, 3 and 4). Heat challenge and tetanic stimulation
At the neuromuscular junction of the frog, a brief period of heating is soon followed by an increase in the frequency of all spontaneous events and an increase in the percentage of subminiatures.15 We repeated this experiment in the electric organ and anaiysed the spontaneous events before, 2 min after and 20 min after a heat challenge at 30°C for 3 min. Changes similar to those reported in frog were observed in three different experiments (Fig. 4). Before heating, the subminiatures were scarce. Two minutes after the challenge, the overall mean frequency rose From about 5 Hz to about 40 Hz and the subminiatures became very numerous. Later, the frequency decreased and the bell-distributed miniatures again represented the major population. Electrical stimulation of the nerves to the electric organ at 10 Hz for 3 min causes a marked “fatigue”
of transmission, which is accompanied by exhaustion of the ATP and creatine phosphate stores of the tissue.’ We recorded spontaneous potentials during the t-10 min which followed such stimulation and found, similarly, that the proportion of subminiatures greatly increased during that period; it reached approximately half of the total number of events (Table I). Poisoned synapses
The above results suggested that the relative increase in subminiatures might in some way be related to the depletion of the energy stores in the tissue. We therefore decided to test the effects of metabolic inhibitors on the preparation. In the presence of dinitrophenol (DNP; 0.2mMf an uncoupler of oxidative phosphorylation, evoked transmitter release was blocked in 3-5 h. At this stage, the acetylcholine (ACh) content was not aitered, but there was a significant reduction in the amount of ATP and a more pronounced decrease in that of creatine phosphate. No significant difference in the frequency of spontaneous potentials was observed with DNP, but the proportion of subminiatures progressively increased during the course of DNP action (Table 1). With a higher concentration of DNP (0.5 mM), the block of transmission occurred more rapidly, the frequency of spontaneous events clearly increased, and a majority of them were also subminiatures. This increase in frequency was independent of the presence of Ca2+ in the bathing medium. Fluorodinitrobenzene (FDNB) is a classical inhibitor of creatine phosphokinase, an enzyme required for the formation of ATP from creatine phosphate and ADP. At a concentration of 0.2 mM, FDNB blocked transmission within 2-3 h and, during the same time, creatine phosphokinase was inhibited by more than 90% (unpublished obser-
Miniature and subminiature potentials in electric organ
919
release. Several observations support the latter explanation. First, the time course of subminiatures was shorter than that of miniatures and for both populations the time course versus amplitude reIayionships followed the same monotonic slope. If they were due to the release of normal sized quanta from the same ele~troplaque but at a distance of the electrode or from eiectrocytes situated underneath, their time course would have been expected to be longer and their time course versus amplitude relationship would have been different. This was actually observed in the few circumstances when two popuIations were clearly present (see Fig. 2). The same change in time course would also have occurred if the subminiatures were due to the release of normal sized quanta at sites distant from the postsynapti~ receptors, for example from nerve endings at sites opposite to the synaptic cleft.” Experiments at the neuromuscular junction where several electrodes were used to explore the releasing sites along the nerve terminal also led to the conclusion that miniatures and subminiatures are produced at the same place. 26~.10 Secondly, in the experiment where the amplitude distributions of spontaneous events during periods of low and high frequency occurrence were compared (Fig. 3), no change in the ratio of miniatures to subminiatures was observed. This is consistent with the view that both miniatures and subminiatures were generated by the same nerve ending ramification. Finally, the experiments where the normal class of miniatures vanished, while the subDISCUSSION miniatures persisted, are difficult to explain if the subminiatures were generated by the release of norThe electric organ in Torpedo is embryologically homologous to neuromuscular systems, but it differs mal sized quanta far from the electrode. From amplitude histograms showing multiple from them in its cytological organization. In the peaks in both the miniature and subminiature popuelectric organ, the nerve endings form an extremely lations, it has been proposed that both classes are ramified network and their presynaptic membrane built up of an integral number of a common small does not show the “active zones” that characterize subunit, the amplitude of which corresponds to the the presumed sites of release in motor nerve termismallest peak of the subminiature population.‘5,‘7,29 nals.2 Also the synaptic vesicles in the Torpedo are While this view has been ~halleng~d,~’ careful statistitwice as large as in the neuromuscular junction. cal analyses has supported the hypothesis of subDespite these structural differences, the electrophysiological aspects of transmitter release in the two units.‘s~‘9The subminiature populations in the present work often displayed multiple peaks (see Fig. 3 and systems have been found to be quite similar. The 5). In some cases, these multiple peaks were also occurrence of spontaneous miniature potentials, the quanta1 composition of the evoked response, and the apparent in the population of bell-distributed miniacalculated number of acetylcholine molecules in a tures (see Figs 1, 3 and 4). However, we did not quantum are the same in electric organ and at the attempt to look for them quantitatively and systemendplate. The present work adds a new similarity: atically, since good stability of the recording condithe demonstration in the Torpedo of a class of small tions is difficult to obtain for a long time with loose minialure potentials resembling in all aspects those patch electrodes. A slight decrease in the negative described in amphibian, mammalian (including man) pressure was able to change the recording conditions. and invertebrate neuromuscular junctions.‘“‘9*2’J7..29 Also the number of recorded events, which is critical The finding was not unexpected, however, since for observing multiple peaks, was possibly not subminiatures have been observed at the sufIicient in all cases. nerve-electroplaque junction of the skate.” Manoeuvres which are known to increase the An important question is whether the subminiature proportion of subminiatures at the neuromuscular potentials are produced by the release of normal sized synapses were also effective in the electric organ. quanta at sites distant from the recording electrode or Subminiatures were abundant during the period folwhether they actually reflect a different type of ACh lowing a heat challenge or a tetanic stimulation, vations). The ATP and ACh contents were greatly decreased by FDNB, but not the amount of creatine pbosphate, which was even increased (Table 1). This is as would be expected since creatine phosphokinase was inactivated. During incubation in the presence of FDNB, the frequency of spontaneous potentials increased by a factor of 10 or 20 and their size distribution changed (Fig. 5). At the time when transmission failed, subminiatures represented 80-90% of the total number of events, and only a few miniatures were still present. These miniatures were of the usual amplitude and time course, which indicated that FDNB did not significantly alter postsynaptic function at this stage {Fig. 5~). The increase in frequency of spontaneous events provoked by FDNB was also observed in the absence of calcium in the bathing medium [solution with I mM ethyleneglycolbis (aminoethylether)tetraacetate and no added calcium]. Table 1 summarizes the results and indicates that the ATP and creatine phosphate contents of the tissue were altered not only with these metabolic poisons, but also in all conditions where we had observed a greater proportion of subminiature potentials, i.e. 24-28 h after excision of the tissue, after a heat challenge or stimulation to exhaustion. These results suggest therefore that the metabolic state of the nerve endings might be important in determining the proportion of subm~niatures among the population of spontaneous events.
920
D. MULLEK and Y. DUNANT
during deterioration of nerve terminals by denervation or by keeping the tissue in Gtro (which also implies a denervation) and under the action of various drugs and toxins such as botulinurn toxin.5.23To test whether alterations of the subminiature to miniature ratio might be linked to changes in either the ACh content or the metabolic state of the tissue, we also analysed the effects of metabolic inhibitors, DNP and FDNB, and determined in all these conditions the ACh, ATP and creatine phosphate contents of the tissue. The results (Table 1) suggest that there was no relationship between the ACh content and the relative number of subminiatures. On the other hand, a better correlation was found with the amount of ATP and creatine phosphate in the tissue. The proportion of subminiatures, in ail conditions tested, seemed to increase proportionately with a reduction in the level of energy rich compounds. This was also true in presence of FDNB, where creatine kinase was inactivated and creatine phosphate could not be used to form ATP, although its concentration in the tissue remains high. Futhermore, in the presence of botulinum toxin, which selectively blocks choiinergic transmission and the occurrence of miniature potentials but not of subminiatures, the contents of ATP and creatine phosphate, but not of ACh. has also recently been found to be reduced.’ It should be kept in mind, however. that the ATP and creatine phosphate measurements were obtained from prisms of electric organ, which include electrocytes and glial ceils in addition to the nerve terminals. Nevertheless, these measurements should at least partly reflect presynaptic changes, since previous work has shown that, during nerve stimulation, about 25% of the energy rich compounds (ATP and creatine phos-
phate) of the tissue are consumed by the nerves endings.’ A number of different hypotheses have been proposed to account for the occurrence of subminiature potentials at the neuromuscular junction (for example: exocytosis of one synaptic vesicle for one subminiature and of several vesicles for one classical miniature, or a partly filled vesicle for a subminiature. or also mechanisms other than vesicle exocytosis, see Ref. 26). In the Torpedo electric organ, biochemical and morphologi~i experiments have suggested that the releasing mechanism might consist of a protein structure inserted in the presynaptic membrane, uses preferentially cytopiastic acetylwhich choiine.“,h.K.Y.i? We propose that this structure might be composed of subunits, which in case of metabolic depletion are activated individually rather than synchronously, so explaining the greater proportion of subminiatures observed in those cases.3 This is, of course, only one possibility and much work will still be needed to determine whether the correlation observed in the present work between the metabolic state of the nerve endings and the proportion of subminiatures results from direct or indirect interactions. In this respect, it will doubtless be fruitful to investigate the biochemical and structural counterparts of these s~lbminiatures in the torpedo electric organ.
.4ckno~I~~~~~zents-- We
thank F. Lo&n for skilful technical assistance, N. Co&t for typing the text and Dr J. Coles for useful suggestions and criticism. This work was supported by the Fonds National Suisse pour la Recherche scientifique (Grant No. X583.0.84) and the Sandoz Foundation.
REFERENCES 1. Chmouliovsky M., Dunant Y. and Hojvat S. (1974)Pre- and postsynaptic utilization of ATP and creatine phosphate at the nerve~le~troplaque junction. J. ~euroche~. 22, 73-75. 2. Couteaux R. and P&cot-Dechavassine M. (1970) V&icules synaptiques et poches au niveau des “zones actives” de la jonction neuromusculaire. C. R. Acad. Sri. Paris 271, 2346-2349. - Dunant Y. (1986) On the mechanism of acetylcholine release. Prog. Neurobiol. 26, 55-92. 4. Dunant Y.. Eder L. and Servetiadis-Hirt L. (1980) Acetylcholine release evoked by single or a few nerve impulses in the electric organ of Torpedo. J. Ph.ysioi., Load. 298, 185-203. 5. Dunant Y., Esquerda J. E., Lo&n F., Marsal J. and Muller D. (In press) Botulinurn toxin blocks quanta1 acetylcholine release and inhibits creatine kinase in the Torpedo electric organ. f. Physiol. 6. Dunant Y., Gautron J., IsraG M., Lesbats B. and Manaranche R. (1972) Les compartiments d’acitylcholine de I’organe blectrique de la Torpille et leurs modifications par la stimulation. J. Neurochem. 19, 1987-2002. 7. Dunant Y. and Muller D. (1986) Quanta1 release of acetylcholine evoked by focal depolarisation at the Torpedo nerve~le~troplaque junction. J. Physiol., Lomd. 379, 461478. 8. Dunant Y., Muller D., ParduEz A.. Jones G. J. and Garcia-Segura L. M. (1984) Augmentation t&s br&ve du nombre de aarticules dans la membrane .prtsynaptiaue . . _ -pendant la transmission d’un influx nerveux. C. R. Acad. Sci. Paris 299, 543-552. 9. Garcia-Segura L. M., Muller D. and Dunant Y. (1986) Increase in the number of presynaptic large intramembrane particles during synaptic transmission at the Torpedo nerve~lectroplaque junction. ~euroscienee 19, 63-79. 10. Gross C. E. and Kriebel M. E. (1983) Skate electric organs generate two classes of spontaneous miniature electrocyte junction potentials. Ahs. &r. Neurosci. 9, 882. Il. Israii M. and Lesbats B. (1981) Continuous determination by a chemiluminescent method of acetylcholine release and compartmentation in Torpen’rielectric organ synaptosomes. J. ;~e~~ocbe~?, 37, 1475-1483. 12. IsraEl M., Manaranche R., Morel N., Dedieu J. C., Gu~ik-Krzyw~cki T. and Lesbats B. (1981) Redistribution of intramembrane particles related to acetylcholine release by cholinergic synaptosomes. J. Ultrmir. Rw. 75, 162. 178. 13. Katz B. (1969) The Release qj’ Neural Transmitter Substances. University Press, Liverpool. 14‘ Kriebel M. E. and Florey E. (1983) Effect of lanthanum ions on the amplitude distribution of miniature endplate potentials and on synaptic vesicles in frog neuromuscular junctions. Neurcl.vcience9, 535-547.
Miniature
and subminiature
potentials
in electric
organ
921
15. Kriebel M. E. and Gross C. E. (1974) Multimodal distribution of frog miniature endplate potentials in adult, denervated and tadpole leg muscle. J. gen. Physiol. 64, 85-103. 16. Kriebel M. E., Hanna R. B. and Pappas G. D. (1980) Spontaneous potentials and fine structure of identified frog denervated neuromuscular junctions. Neuroscience 5, 97-108. 17. Kriebel M. E., Llados F. and Matteson D. R. (1976) Spontaneous subminiature endplate potentials in mouse diaphragm muscle: evidence for synchronous release. J. Physiol., Lond. 262, 553-581. 18. Kriebel M. E., Llados F. and Matteson D. R. (1982) Histograms of the unitary evoked potential of the mouse diaphragm show multiple peaks. J. Physiol., Lond. 322, 21 I-222. 19. Matteson D. R., Kriebel M. E. and Llados F. (1981) A statistical model indicates that miniature end-plate potentials and unitary evoked end-plate potentials are composed of subunits. /. theor. Rio/. 90, 337-363. 20. Miledi R., Mohnoff P. and Potter L. T. (1971) Isolation of the cholinergic receptor protein of Torpedo electric tissue. Nature, Land. 229, 554-557. 21. Miller D. C., Weinstock M. M. and Magleby K. L. (1978) Is the quantum of transmitter release composed of subunits? Nature, Lond. 274, 388-390. 22. Muller D. (1986) Potentiation by 4-aminopyridine of quanta1 acetylcholine release at the Torpedo nerveeelectroplacque junction. J. Physiol., Lond. 379, 461478. 23. Muller D. and Dunant Y. (1985) Subminiature electroplaque potentials are present in Torpedo electric organ. Experientia 41, 824. 24. Soria B. (1983) Properties of miniature post-synaptic currents at the Torpedo rnarrnorafa nerveelectroplaque junction. Quarf. J. exp. Physiol. 68, 189-202. 25. Strehler B. L. (1974) Adenosine-5’-triphosphate and creatine phosphate. determination with luciferase. In Methods in Enzymatic Analysis, Vol. 4, pp. 2112-2115. Academic Press, New York. 26. Tremblay J. P., Laurie R. E. and Colonnier M. (1983) Is the MEPP due to the release of one vesicle or to similtaneous release of several vesicles at one active zone? Brain Res. Rev. 6, 299-314. 27. Vautrin J. and Mambrini J. (1981) Caracteristiques du potentiel unitaire de plaque motrice de la grenouille. J. Physial., Paris 77, 999-1010. 28. Walther C., Hodgkiss J. P. and Hensler K. (1982) Sub-miniature junction potentials in excitatory neuromuscular synapses of the locust. Neurosci. Lett. 34, 27-32. 29. Wernig A. and Stirner H. (1977) Q uantum amplitude distributions point to functional unity of the synaptic “active zone”. Nature, Lond. 268, 82&822. 30. Wernig A. and Motelica-Heino 1. (1978) On the presynaptic nature of the quanta1 subunits. Neurosci. Let/. 8, 231k234.
(Accepfed 5 August 1986)