Gen Pharmac Vol 23, No 2, pp 165-169, 1992 Planted m Great Britain All rights reserved
0306-3623/92 $5 00 + 0 00 Copyright © 1992 Pergamon Press pie
DIVERSITY AMONG MOUSE MOTOR NERVE TERMINALS WITH RESPECT TO RELEASE TRANSMITTER QUANTA MASAKAZUNISHIMURA,I* KIKUKO TSUBAKI2 and OSAMU YAGASAKI2 Departments of tToxlcology and 2pharmacology, Umverslty of Osaka Prefecture School of Vetennary Medicine, Sakal, Osaka 591, Japan (Reeewed 30 July 1991) Abstract--1 The aim of this work was to reexamine whether a posmve correlation exists between the frequency (F, sec- ]) of mlmature endplate potentials (me p ps) and the quantal content (m) of endplate potenUals (e p ps) or between quantal content, frequency and twin-pulse facd~tatlon of transmitter release at a large number of neuromuscular junctions m the mouse 2 The values of F and m were both measured mtracellulary at endplates of mouse diaphragm m a high Mg 2+/low Ca 2+ bathing solution 3 Values of both F and m vaned from junction to junction Smaller values of F were correlated with smaller values of m, and wce versa, resulting m a hnear relationships Histograms of F and m were skewed towards smaller values 4 E p ps evoked by twin pulses gave the quantal contents of the first (m 1) and second (m2) responses 5 The ratm of m 2 to m 1 vaned from junctmn to junction A histogram of this ratm was skewed towards smaller values 6 The rauo of m2 to m 1 showed larger fluctuations at junctmns with smaller values of F or m 1 but was focused around 1 at juncuons with larger values of F or m 1 7 The skewed parts of the histograms of F, m and m2/ml accounted for the major population of junctions 8 These results support the hypothests that an intrinsic abfltty to release transmitter plays a role m regulatmn of the evoked output of transmitter at neuromuscular junctions m the mouse 9 Such an ablhty ~s not correlated w~th the twin-pulse facfl~tatmn of transnutter release
INTRODUCTION
There are reports, based exclusively on qualltahve results, that (1) higher spontaneous acUvlty is probably correlated with higher rates of evoked release of transmitter, when both spontaneous and evoked release of transmitter are considered at the same neuromuscular junction (Grmnell and Pawson, 1989, Rahamlmmof, 1976), and that (2) m control preparations, an increase m mtracellular levels of Ca 2+ runs augments both the spontaneous and evoked released of transmitter ( R a h a m l m m o f et al., 1978). By contrast, the results of Propst and Ko (Propst and Ko, 1987), obtained from relatively few samples, indicate that a highly sigmficant positive correlation exists between the area of the active zone (A) and evoked release but not between A and the frequency of m.e.p ps. Thus, their results imply that synaptlc structures of the membrane responsible for the regulatmn of both modes of transmitter release may differ from each other If a positive correlatmn exists between the rates of both modes of transmitter release (Gnnnell and Pawson, 1989; Raharmmmof, 1976), even though the synaptic structures of the membrane differ with respect to regulation of the two modes (Propst and Ko, 1987), it is possible that the correlation is inde-
*To whom all correspondence should be addressed OP 23/2--B
pendent of the difference m the synapUc structures of the membrane Since a w~de v a n a t m n extsts In the frequency of m e p ps (Erulkar and Rahanummof, 1976) as well as m quantal content (Gnnnell and Herrera, 1980), it should be possible to examine the correlatmn between both modes of transmitter release at a large number of neuromuscular junctions If it exists, such a correlaUon may also be reflected tn the ability of nerve terminals to alter the rate of release of transmitter, for instance, by temporal facfl~tauon The present study was performed to reexamine whether a p o m i v e correlatmn exists between the frequency of m e p.ps (F) and quantal content (m) or between these parameters and twin-pulse facd~tation of transmitter release at a large number of mouse neuromuscular junctions MATERIALS AND M E T H O D S
Expenments were performed on preparations of tsolated left hermdlaphragm from male ddY mice of 9 to 12 weeks of age The preparation was pruned to a slhcone resin, which hned the bottom of a plasttc chamber with a capacity of about 30 ml, and was soaked m Krebs-lhnger solution. The solution was constantly reorculated by means of an "oxygen hft" system The circulating soluUon had the following composition (mM): NaCI, 135, KC1, 5; CaCI2, 2; MgCI2, 1; NaHCO3, 15; glucose, 11. A bathing solution which contained 5 mM Mg2÷ was prepared, and the solution was made 0 4 to 0.8 milhmolar in Ca 2+ ions, for measurements 165
MASAKAZU NISHIMURA el al
166
of e p ps The bathing solution was bubbled wxth a mixture of 95% 02 and 5% CO2 and maintained at pH 7 3 and 36°C The temperature of the solution m the bath was monitored by a thermlster (model MGA-II, Shlbaura Electnc Co, Tokyo) and held constant by means of an external water jacket and a thermoregulatory dewce (Thermomlnder Mlm 80, Tmyo, Tokyo) during each expenment Intracellular recordings were made with glass torerocapillary electrodes, filled with 3M KC1, each with resistance of 5 to 6 MQ The electrode was inserted into fibres near endplate regions The signals were led through a hlghqmpedance, unity-gain preamphfier (MEZ-8201, Nlhon Kohden, Tokyo) m an oscilloscope (VC-10, Nihon Kohden), and stored on an FM instrumentation tape recorder (RMG-5204, Nihon Kohden) when necessary Preparations were exposed to the test bathing solution for 20 rain before measurements of potentml changes At each endplate, m e p ps were recorded for about 1 mm at first, then the nerve trunk was acUvated The measurements were terminated within 1 5 hr from the start of the expenment Single e p ps were evoked by stimulation of the phremc nerve with 128 or 256 square pulses of 0 1 msec in duratmn and a supramaxlmal voltage of 1 Hz, by use of a sucUon electrode in a bathing solution with 5ram Mg2+ that contained vanous concentratxons, [Ca 2+]o, of Ca 2+ ions In other experiments, the nerves were electrically condmoned to change their neuronal reactmty Pmred pulses with intervals 2 or 4 ms were given 128 or 256 times at intervals of 1 5 sec in a bathing solutzon with 5raM Mg 2+ that contained 0 6 mM Ca 2+, after measurement of m e p ps for about 1 mln This tnal resulted m e p ps of the first and second responses The quantal content was estimated by the method of failures from the formula m = loge(U/No),
where N is the number of trials and N O IS the number of failures (Crawford, 1974) Student's t-test were used for stat=stlcal analyses and a probablhty of less than 0 05 was deemed statistically s~gmficant All the chemicals used were of analytzcal grade
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F Fig 1 The relatmnshlp between the frequency (F, sec -~ ) of m e p ps and the quantal content (m) of e p ps at mouse neuromuscular junctions in a bathing solution w~th 5 mM Mg 2+ that contained 0 6 mM Ca 2+ Measurements were obtained at 102 end-plates of 5 preparatmns (A, each symbol corresponds to an lndmdual preparatmn) and at 584 end-plates of 26 preparauons (B) Both slopes of the relationship of F and m were slgmficant (P < 0 05)
mg o n the terminals, and that such d~verslty is c o m m o n to b o t h resting and evoked actzwt~es Relationships between F and m were plotted and h~stograms constructed with other data obtained at
RESULTS 90
The frequency (F, sec - l ) o f m e.p.ps and the quantal content (m) o f e p.ps were b o t h measured at the same endplates o f preparaUons o f mouse dmp h r a g m s m a bathing soluuon wzth 5 r a M M g 2+ that contained various c o n c e n t r a t m n s o f Ca 2 + ions Fzgure 1 shows the relationship between F and m at 0 6 m M [Ca2+]o The upper panel (A) is an mdlvlduahzed presentation o f measurements d e n v e d f r o m different preparations, indicated by dzfferent symbols In each o f five preparations, there seems to be a correlation between values o f F and rn; smaller values o f F c o r r e s p o n d to smaller values o f m and r a c e v e r s a Such a relatzonshzp held m m a n y m o r e measurements from a total o f 26 preparatzons (B) M o s t determinations were conducted within 1 to 5 sec-1 for F and within 0 1 to 0 4 for rn However, at some juncttons, m i n o r c o m p o n e n t s generated m u c h higher values for b o t h parameters. Spontaneous actzwty, being indicated as F, correlated well with evoked output o f transmztter, generaUng a regression hne with a slgmficant slope ( P < 0.05). Figure 2 shows hzstograms o f values o f F and rn. Values o f F are concentrated at lower values, thereby producing an asymmetric dzstributlon pattern. Such a profile is also f o u n d m the histogram o f the quantal content (m) o f e p ps. These data indicate that rates o f release o f transmitter are heterogeneous, depend-
F
A
H=stogramof F
.¢•60 30
0 0
4
8
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' ' '1'2 . . . . . . .
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Histogram of m
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=.- 80 ®
0
,,.,
. . . . . . . .0. 4
, . . .0. .8. .
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Fig 2 (A) Histograms of the frequency (F, sec -I) of m e p ps prepared from the data used to generate Fig IB (B) Histograms of the quantal content (m) of e p ps prepared from the data used to generate F~g I B Note the skewed dlstnbuUon
167
~iverslty among motor nerve termlnals
A
B
J
Fig 3 Examples of m e p ps and e p ps evoked by stlmulaUon with twin pulses with 4 msee intervals of the dmphragm muscle of a mouse All the measurements were obtained from the same end-plate (A) M e p ps recorded m a bathing solutmn with 5 mM Mg 2+ that contained 0 6 mM Ca 2+ (supenmposed tracings from 5 sweeps) (B) E p ps recorded m the same solutaon as m (A) (Supenmposed tracing of 5 sweeps) Resting membrane potentmls were between 70 and 76 mV Callbralaon 2 msec, 1 mV [Ca2+]o of 0 4 and 0 8 m M Such trials gave very similar results at both values of [Ca2+]o In the second tnal, twin pulses vath intervals of 2 or 4 msec were given to the nerve trunk at intervals of 1 5 see m a bathing solution with 5 m M M g 2÷ that contained vanous concentrations of Ca 2+ ions The nerve trunk was activated 256 times at [Ca2+]o of 0.4 and 0 . 6 m M and 128 times at [Ca2+]o of 0 . 8 m M . Typical results, w~th spontaneous potential changes, can be seen in Fig 3 Potentml changes occurred spontaneously with amplitudes of 0 1 mV or more and rise times of 1 msec or less at each endplate, being m e.p ps M e p ps were recorded prior to stimulation o f the nerve trunk at an endplate Stimulation o f nerve trunks w~th twin pulses, with intervals of 4 msec m this case, gave endplate responses with amplitude and rise times slmdar to those of m e p.ps F r o m results of many trmls ~t was clear that the frequency (F, sec- t) of m e p ps and the quantal contents of the first (m l) and second (m2) responses vaned from fiber to fiber At endplates voth smaller values o f F, values of m 1 and m 2 were also smaller, and wce versa, as shown in Fig 1, generating regression lines voth s~gnlficant slopes (P < 0 05, data not shown) Most measured values were concentrated around about 3 sec -1 for F and around 0 2 for m l and m2, respectively, for example at [Ca 2÷ ]o of 0 6 m M Thus, the histograms for F, m 1 and m2 showed skewed patterns similar to those shown in Fig 2, indicating the presence of significant numbers of components with larger values of F, m l and m2 Very simdar results were obtained in the other case of stimulation by twin pulses with 2 msec intervals. As mentioned, the twin pulses gave the quantal contents of the first (m 1) and second (m2) responses. The mean value of m 2 was significantly larger than that o f m 1 for the paired pulses at both intervals The larger value for m2 indicates an increase in the amount of transmitter released at the second Impulse. This phenomenon is known as the twin-pulse faclhration of transmitter release It results from condltloning by the first stimulus The ratio of m2 to m 1 can be taken as an indicator of the extent of the facilitation. Averaged values for the ratao of m2 to m l were similar, Irrespective o f the intervals of the pmred pulses Indivadual values of the ratm of m 2 to m 1 vaned widely If the abdlty of the nerve terminal
to release transmitter is heterogeneous, it ~s possible that such a charactenstIc may be reflected In the altered state, with respect to release of transmitter, as facdItaUon Figure 4 shows histograms o f the ratm o f m2 to m 1 when nerve trunks were stimulated with twm pulses, with mtervals of 4 msec in this case, m a bathing solution vath 5 m M M g 2+ that contained 0.6raM Ca 2+ ions. The values of m2/ml were skewed in the direction of lower values Smce the ratio of m2 to m l reflects the abdlty of nerve terminals to respond to c o n d m o m n g shocks and smce this abihty may reflect the resting abd~ty to release transnutter, relatmnslups were exammed between the parameters F, m 1 and m2/m 1 obtamed by twin-pulse stlmulatmn with 4 msec mtervals. F~gure 5 shows the relatlonslups between m2/m I and F and between m2/m 1 and m 1. In both cases, the chstnbutaon of values of m2/m 1 is skewed to one side, as was seen in Fig. 4. Also m both cases, the chstributmn o f the ratao of m 2 to m I fluctuated more when values o f F or m 1 were small; but there was a gradual dechne m the fluctuatmn with increases m the values of F or m 1. The ratio was finally concentrated around 1 when values of F or m 1 were largest Similar results were obtamed m the other cases of stimulation by won pulses with 2 msec mtervals.
H~togram of rn2/ml
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m2/ml Fzg 4 Histogram of values of twm-pulse faafitaUon (the ratio of m2/ml) of transmitter release The parameter m2/ml was obtamed by stnnulation with tvan pulses with a 4 msec interval at mouse neuromuscular junctions in a bathmg solution vath 5 mM Mg2+ that contamed 0 6 mM Ca 2+ ions Number of observations 391 end-plates m 17 preparations Note the skewed distnbutlon
168
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Fig 5 Relationship between the twin-pulse facilitation (the ratio of m2 to m 1) of transmitter release and the frequency (F, sec -I) of m e p ps (A) and between the ratio of m2 to m 1 and the first quantal content (m I) of e p ps (B) Data were the same as those used to generate F~g 4 DISCUSSION Propst and Ko (Propst and Ko, 1987) indicated that, m frog neuromuscular junctions, the active zone plays a role m regulating the evoked release of transmatter but is not significantly involved in regulating the frequency of m e p ps They suggested that this difference rmght be due to &fferent processes or synaptic structures being responsible for the regulation of the two modes of release transmatter Our experiments indicate that there can be large variations m e~ther the frequency of m e p ps or the quantal content of e p ps at a large number of mouse neuromuscular junctions and that the variations are parallel between them These observations confirm results reported by other investigators (Kuno et al, 1971, R a h a m i m o f et al, 1978) Thus, It Is posmble that pre-membrane process(es) may regulate both modes of release of transmatter In common, even though the structural process associated with the plasma membrane &fferentmtes between the two modes (Propst and Ko, 1987) The rate of spontaneous release of transmitter quanta is shown as the frequency of occurrence of m.e p ps The frequency is less dependent on [Ca2+]o than is the quantal content of e p ps (Matthews and Wickelgren, 1977) However, an elevation of [Ca 2 + ], by means of injection of Ca 2+ Ions into nerve terminals (Mde&, 1973; R a h a m l m o f et al, 1978) or by m h i b m o n of matochondnal function (Alnaes and Rahamimoff, 1975, R a h a m l m o f f and Alnaes, 1973) causes a major increase in the frequency of m e p ps Thus, it is clear that the frequency ofm.e.p.ps reflects the availability of [Ca2+], at the resting state, as suggested by many investigators The many fluctuatmns in this rate suggest the probable &vermty of nerve terminals in terms of their abihty to maintain a particular [Ca:+]~.
el
a/
The evoked release of transmitter quanta depends on [Ca2+]o (Katz, 1969) Internal Ca 2÷ ions present at rest act in concert with Ca 2+ ions that have newly entered the terminal after a nerve impulse (Katz, 1969) This summation of Ca 2+ ions leads to elevation of [Ca 2+]1 and a larger quantal content of e p ps (Alnaes and Rahamlmoff, 1975) Thus, the evoked release probably reflects not only the permeablhty of the nerve-terminal membrane to Ca 2+ ions but also the resting availability of residual Ca 2+ ions inside the terminal This possibility is supported by previous (Grinnel and Pawson, 1989, Rahamlmoff, 1976, R a h a m l m o f f e t al, 1978)results and ours, which showed that the two modes of transmitter release were correlated with each other These results lead to a hypothesis that the diversity of releasablllty at premembrane process(es) is c o m m o n to both modes of release of transmitter quanta If a series of two, closely spaced, nerve impulses are given to the nerve trunk, the second response is larger than the first (Wernlg, 1972, Zucker, 1973) This phenomenon is called as temporal faclhtatlon (Del Castillo and Katz, 1954) The faclhtaUon can be quantltated as the ratio of m2 to m 1 (Del Castlllo and Katz, 1954). This ratio grows as the interval between the twin shocks is reduced (Mallart and Martin, 1968) This phenomenon has been interpreted m terms of the "remdual calcmm" hypothesis (Katz and Mde&, 1968, Rahamlmoff, 1968) It reflects the residual availability of internal Ca 2 + ions, the level which is elevated by the first nerve impulse The residual internal Ca 2 + act with newly available Ca 2+ ions, introduced by the second impulse, leading to an increased rate of transmitter release The ratm of m2 and m 1 is dependent on the quantal content of the first e p p (ml), the higher is the value o f m l the smaller is the ratio of m 2 to m I (Mallart and Martin, 1968) This result held true in the present experiments Furthermore, such a relationship was also observed between the ratio of m2 and rn 1 and the frequency of m e p ps, which probably reflected the residual availability of internal Ca 2+ ions Thus, heterogeneity was also noted in the &stnbutlon of the ratio of m2 to m l However, no parallelism was demonstrated between the ratio of m 2 to m 1 and m 1 or the frequency of m e p ps, perhaps due to the fact that the increase in the ratio of m2 to m l Is an excitatory phenomenon on the mllhsecond scale The first e p p is evoked separately at several seconds and the m e p p reflects the resting condition In summary, even if the structural step assocmted with the membrane of the nerve terminal &fferentlates between spontaneous and evoked release of transmitter quanta (Propst and Ko, 1987), pre-membrane process(es) may regulate both modes of release of transmitter in c o m m o n Thin pre-membrane process(es) does not seem to include the mechamsm for sequestering internal Ca 2+ Ions, which Is suggested to functmn in the temporal facdltatlon of the release of transmitter quanta REFERENCES Alnaes E. and Rahamlmoff R (1975) On the role of mttochondna in transmitter release from motor nerve terminals J. Physiol 248, 285-306
Diversity among motor nerve terminals del Castlllo J and Katz B (1954) Statistical factors Involved m neuromuscular facilitation and depression J Physwl 124, 574-585 Crawford A C (1974) The dependence of evoked transtarter release on external calcium tons at very low mean quantal contents J Phystol 240, 255-278 Erulkar S D and Rahamlmoff R (1976) Peno&c fluctuations m transmitter release at the frog neuromuscular junction J Physwl 256, 20-21P Gnnnell A D and Herrera A A (1980) Physiological regulation of synaptm effectiveness at frog neuromuscular junction J Physwl 307, 301-317 Gnnnell A D and Pawson P A (1989) Dependence of spontaneous release at frog junctions on synaptm strength, external calcium and terminal length J Physwl 418, 397-410 Katz B (1969) The Release of Neural Transmztter Substances pp 1-55 Charles C Thomas, Springfield, Illinois Katz B and Mlledl R (1968) The role of calcmm in neuromuscular faclhtaUon J Physwl 195, 481-492 Kuno M , Turkams S A and Weakly J N (1971) Correlation between nerve terminal size and transmitter release at the neuromuscular junction of the frog J Phystol 213, 545-556 MaUart A and Martin A R (1968) The relation between quantal content and facilitation at the neuromuscular junction of the frog J Physwl 195, 593-604 Matthews G and Wlckelgren W O (1977) On the effect of calcium on frequency of miniature end plate potentials at the frog neuromuscular junction J Physwl 226, 91-101
169
Mde& R (1973) Transmitter release reduced by rejection of calcmm ions into nerve terminals Proc. Roy Soc, B 183, 421-425 Propst J W and Ko C P (1987) Correlation between acuve zone ultrastructure and synaptlc function studied with freeze-fracture of physiologically identified neuromuscular Junctions J, Neuroscz 7, 3654-3664 Rahamlmoff R (1968) A dual effect of calcium ions on neuromuscular faclhtatlon J Physwl 195, 471--480 Rahamlmoff R (1976) The role of calcium m transmRter release at the neuromuscular junction In Motor Innervatzon of Muscle (E&ted by Thesleff S), pp 117-149 Academic Press, New York Rahamlmoff R and Alnaes E (1973) Inhibitory action of ruthenium red on neuromuscular transmission Proc natn Acad Sct U S A 70, 3613-1616 Rahamlmoff R , Erulkar S D , Lev-Tov A and Mere H (1978) Intracellular and extracellular calcmm Ions m transmitter release at the neuromuscular synapse Ann New York Acad Sct 583-598 Rahamlmoff R , Mere H , Erulkar S D and Barenholtx Y (1978) Changes in transmitter release induced by ton containing hposomes Proc natn Acad Scl U S A 75, 5214-5216 Wernlg A (1972) Changes in statistical parameters dunng facthtatlon at the crayfish neuromuscular junction J Physwl 226, 751-759 Zucker R S (1973) Changes In the statistics of transmitter release dunng facthtatton J Physwl 229, 787-810
0306-3623/92 $5 00 + 0 00 Copyright © 1992 Pergamon Press pie
DIVERSITY AMONG MOUSE MOTOR NERVE TERMINALS WITH RESPECT TO RELEASE TRANSMITTER QUANTA MASAKAZUNISHIMURA,I* KIKUKO TSUBAKI2 and OSAMU YAGASAKI2 Departments of tToxlcology and 2pharmacology, Umverslty of Osaka Prefecture School of Vetennary Medicine, Sakal, Osaka 591, Japan (Reeewed 30 July 1991) Abstract--1 The aim of this work was to reexamine whether a posmve correlation exists between the frequency (F, sec- ]) of mlmature endplate potentials (me p ps) and the quantal content (m) of endplate potenUals (e p ps) or between quantal content, frequency and twin-pulse facd~tatlon of transmitter release at a large number of neuromuscular junctions m the mouse 2 The values of F and m were both measured mtracellulary at endplates of mouse diaphragm m a high Mg 2+/low Ca 2+ bathing solution 3 Values of both F and m vaned from junction to junction Smaller values of F were correlated with smaller values of m, and wce versa, resulting m a hnear relationships Histograms of F and m were skewed towards smaller values 4 E p ps evoked by twin pulses gave the quantal contents of the first (m 1) and second (m2) responses 5 The ratm of m 2 to m 1 vaned from junctmn to junction A histogram of this ratm was skewed towards smaller values 6 The rauo of m2 to m 1 showed larger fluctuations at junctmns with smaller values of F or m 1 but was focused around 1 at juncuons with larger values of F or m 1 7 The skewed parts of the histograms of F, m and m2/ml accounted for the major population of junctions 8 These results support the hypothests that an intrinsic abfltty to release transmitter plays a role m regulatmn of the evoked output of transmitter at neuromuscular junctions m the mouse 9 Such an ablhty ~s not correlated w~th the twin-pulse facfl~tatmn of transnutter release
INTRODUCTION
There are reports, based exclusively on qualltahve results, that (1) higher spontaneous acUvlty is probably correlated with higher rates of evoked release of transmitter, when both spontaneous and evoked release of transmitter are considered at the same neuromuscular junction (Grmnell and Pawson, 1989, Rahamlmmof, 1976), and that (2) m control preparations, an increase m mtracellular levels of Ca 2+ runs augments both the spontaneous and evoked released of transmitter ( R a h a m l m m o f et al., 1978). By contrast, the results of Propst and Ko (Propst and Ko, 1987), obtained from relatively few samples, indicate that a highly sigmficant positive correlation exists between the area of the active zone (A) and evoked release but not between A and the frequency of m.e.p ps. Thus, their results imply that synaptlc structures of the membrane responsible for the regulatmn of both modes of transmitter release may differ from each other If a positive correlatmn exists between the rates of both modes of transmitter release (Gnnnell and Pawson, 1989; Raharmmmof, 1976), even though the synaptic structures of the membrane differ with respect to regulation of the two modes (Propst and Ko, 1987), it is possible that the correlation is inde-
*To whom all correspondence should be addressed OP 23/2--B
pendent of the difference m the synapUc structures of the membrane Since a w~de v a n a t m n extsts In the frequency of m e p ps (Erulkar and Rahanummof, 1976) as well as m quantal content (Gnnnell and Herrera, 1980), it should be possible to examine the correlatmn between both modes of transmitter release at a large number of neuromuscular junctions If it exists, such a correlaUon may also be reflected tn the ability of nerve terminals to alter the rate of release of transmitter, for instance, by temporal facfl~tauon The present study was performed to reexamine whether a p o m i v e correlatmn exists between the frequency of m e p.ps (F) and quantal content (m) or between these parameters and twin-pulse facd~tation of transmitter release at a large number of mouse neuromuscular junctions MATERIALS AND M E T H O D S
Expenments were performed on preparations of tsolated left hermdlaphragm from male ddY mice of 9 to 12 weeks of age The preparation was pruned to a slhcone resin, which hned the bottom of a plasttc chamber with a capacity of about 30 ml, and was soaked m Krebs-lhnger solution. The solution was constantly reorculated by means of an "oxygen hft" system The circulating soluUon had the following composition (mM): NaCI, 135, KC1, 5; CaCI2, 2; MgCI2, 1; NaHCO3, 15; glucose, 11. A bathing solution which contained 5 mM Mg2÷ was prepared, and the solution was made 0 4 to 0.8 milhmolar in Ca 2+ ions, for measurements 165
MASAKAZU NISHIMURA el al
166
of e p ps The bathing solution was bubbled wxth a mixture of 95% 02 and 5% CO2 and maintained at pH 7 3 and 36°C The temperature of the solution m the bath was monitored by a thermlster (model MGA-II, Shlbaura Electnc Co, Tokyo) and held constant by means of an external water jacket and a thermoregulatory dewce (Thermomlnder Mlm 80, Tmyo, Tokyo) during each expenment Intracellular recordings were made with glass torerocapillary electrodes, filled with 3M KC1, each with resistance of 5 to 6 MQ The electrode was inserted into fibres near endplate regions The signals were led through a hlghqmpedance, unity-gain preamphfier (MEZ-8201, Nlhon Kohden, Tokyo) m an oscilloscope (VC-10, Nihon Kohden), and stored on an FM instrumentation tape recorder (RMG-5204, Nihon Kohden) when necessary Preparations were exposed to the test bathing solution for 20 rain before measurements of potentml changes At each endplate, m e p ps were recorded for about 1 mm at first, then the nerve trunk was acUvated The measurements were terminated within 1 5 hr from the start of the expenment Single e p ps were evoked by stimulation of the phremc nerve with 128 or 256 square pulses of 0 1 msec in duratmn and a supramaxlmal voltage of 1 Hz, by use of a sucUon electrode in a bathing solution with 5ram Mg2+ that contained vanous concentratxons, [Ca 2+]o, of Ca 2+ ions In other experiments, the nerves were electrically condmoned to change their neuronal reactmty Pmred pulses with intervals 2 or 4 ms were given 128 or 256 times at intervals of 1 5 sec in a bathing solutzon with 5raM Mg 2+ that contained 0 6 mM Ca 2+, after measurement of m e p ps for about 1 mln This tnal resulted m e p ps of the first and second responses The quantal content was estimated by the method of failures from the formula m = loge(U/No),
where N is the number of trials and N O IS the number of failures (Crawford, 1974) Student's t-test were used for stat=stlcal analyses and a probablhty of less than 0 05 was deemed statistically s~gmficant All the chemicals used were of analytzcal grade
A
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F Fig 1 The relatmnshlp between the frequency (F, sec -~ ) of m e p ps and the quantal content (m) of e p ps at mouse neuromuscular junctions in a bathing solution w~th 5 mM Mg 2+ that contained 0 6 mM Ca 2+ Measurements were obtained at 102 end-plates of 5 preparatmns (A, each symbol corresponds to an lndmdual preparatmn) and at 584 end-plates of 26 preparauons (B) Both slopes of the relationship of F and m were slgmficant (P < 0 05)
mg o n the terminals, and that such d~verslty is c o m m o n to b o t h resting and evoked actzwt~es Relationships between F and m were plotted and h~stograms constructed with other data obtained at
RESULTS 90
The frequency (F, sec - l ) o f m e.p.ps and the quantal content (m) o f e p.ps were b o t h measured at the same endplates o f preparaUons o f mouse dmp h r a g m s m a bathing soluuon wzth 5 r a M M g 2+ that contained various c o n c e n t r a t m n s o f Ca 2 + ions Fzgure 1 shows the relationship between F and m at 0 6 m M [Ca2+]o The upper panel (A) is an mdlvlduahzed presentation o f measurements d e n v e d f r o m different preparations, indicated by dzfferent symbols In each o f five preparations, there seems to be a correlation between values o f F and rn; smaller values o f F c o r r e s p o n d to smaller values o f m and r a c e v e r s a Such a relatzonshzp held m m a n y m o r e measurements from a total o f 26 preparatzons (B) M o s t determinations were conducted within 1 to 5 sec-1 for F and within 0 1 to 0 4 for rn However, at some juncttons, m i n o r c o m p o n e n t s generated m u c h higher values for b o t h parameters. Spontaneous actzwty, being indicated as F, correlated well with evoked output o f transmztter, generaUng a regression hne with a slgmficant slope ( P < 0.05). Figure 2 shows hzstograms o f values o f F and rn. Values o f F are concentrated at lower values, thereby producing an asymmetric dzstributlon pattern. Such a profile is also f o u n d m the histogram o f the quantal content (m) o f e p ps. These data indicate that rates o f release o f transmitter are heterogeneous, depend-
F
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Fig 2 (A) Histograms of the frequency (F, sec -I) of m e p ps prepared from the data used to generate Fig IB (B) Histograms of the quantal content (m) of e p ps prepared from the data used to generate F~g I B Note the skewed dlstnbuUon
167
~iverslty among motor nerve termlnals
A
B
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Fig 3 Examples of m e p ps and e p ps evoked by stlmulaUon with twin pulses with 4 msee intervals of the dmphragm muscle of a mouse All the measurements were obtained from the same end-plate (A) M e p ps recorded m a bathing solutmn with 5 mM Mg 2+ that contained 0 6 mM Ca 2+ (supenmposed tracings from 5 sweeps) (B) E p ps recorded m the same solutaon as m (A) (Supenmposed tracing of 5 sweeps) Resting membrane potentmls were between 70 and 76 mV Callbralaon 2 msec, 1 mV [Ca2+]o of 0 4 and 0 8 m M Such trials gave very similar results at both values of [Ca2+]o In the second tnal, twin pulses vath intervals of 2 or 4 msec were given to the nerve trunk at intervals of 1 5 see m a bathing solution with 5 m M M g 2÷ that contained vanous concentrations of Ca 2+ ions The nerve trunk was activated 256 times at [Ca2+]o of 0.4 and 0 . 6 m M and 128 times at [Ca2+]o of 0 . 8 m M . Typical results, w~th spontaneous potential changes, can be seen in Fig 3 Potentml changes occurred spontaneously with amplitudes of 0 1 mV or more and rise times of 1 msec or less at each endplate, being m e.p ps M e p ps were recorded prior to stimulation o f the nerve trunk at an endplate Stimulation o f nerve trunks w~th twin pulses, with intervals of 4 msec m this case, gave endplate responses with amplitude and rise times slmdar to those of m e p.ps F r o m results of many trmls ~t was clear that the frequency (F, sec- t) of m e p ps and the quantal contents of the first (m l) and second (m2) responses vaned from fiber to fiber At endplates voth smaller values o f F, values of m 1 and m 2 were also smaller, and wce versa, as shown in Fig 1, generating regression lines voth s~gnlficant slopes (P < 0 05, data not shown) Most measured values were concentrated around about 3 sec -1 for F and around 0 2 for m l and m2, respectively, for example at [Ca 2÷ ]o of 0 6 m M Thus, the histograms for F, m 1 and m2 showed skewed patterns similar to those shown in Fig 2, indicating the presence of significant numbers of components with larger values of F, m l and m2 Very simdar results were obtained in the other case of stimulation by twin pulses with 2 msec intervals. As mentioned, the twin pulses gave the quantal contents of the first (m 1) and second (m2) responses. The mean value of m 2 was significantly larger than that o f m 1 for the paired pulses at both intervals The larger value for m2 indicates an increase in the amount of transmitter released at the second Impulse. This phenomenon is known as the twin-pulse faclhration of transmitter release It results from condltloning by the first stimulus The ratio of m2 to m 1 can be taken as an indicator of the extent of the facilitation. Averaged values for the ratao of m2 to m l were similar, Irrespective o f the intervals of the pmred pulses Indivadual values of the ratm of m 2 to m 1 vaned widely If the abdlty of the nerve terminal
to release transmitter is heterogeneous, it ~s possible that such a charactenstIc may be reflected In the altered state, with respect to release of transmitter, as facdItaUon Figure 4 shows histograms o f the ratm o f m2 to m 1 when nerve trunks were stimulated with twm pulses, with mtervals of 4 msec in this case, m a bathing solution vath 5 m M M g 2+ that contained 0.6raM Ca 2+ ions. The values of m2/ml were skewed in the direction of lower values Smce the ratio of m2 to m l reflects the abdlty of nerve terminals to respond to c o n d m o m n g shocks and smce this abihty may reflect the resting abd~ty to release transnutter, relatmnslups were exammed between the parameters F, m 1 and m2/m 1 obtamed by twin-pulse stlmulatmn with 4 msec mtervals. F~gure 5 shows the relatlonslups between m2/m I and F and between m2/m 1 and m 1. In both cases, the chstnbutaon of values of m2/m 1 is skewed to one side, as was seen in Fig. 4. Also m both cases, the chstributmn o f the ratao of m 2 to m I fluctuated more when values o f F or m 1 were small; but there was a gradual dechne m the fluctuatmn with increases m the values of F or m 1. The ratio was finally concentrated around 1 when values of F or m 1 were largest Similar results were obtamed m the other cases of stimulation by won pulses with 2 msec mtervals.
H~togram of rn2/ml
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168
MASAKAZU
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Fig 5 Relationship between the twin-pulse facilitation (the ratio of m2 to m 1) of transmitter release and the frequency (F, sec -I) of m e p ps (A) and between the ratio of m2 to m 1 and the first quantal content (m I) of e p ps (B) Data were the same as those used to generate F~g 4 DISCUSSION Propst and Ko (Propst and Ko, 1987) indicated that, m frog neuromuscular junctions, the active zone plays a role m regulating the evoked release of transmatter but is not significantly involved in regulating the frequency of m e p ps They suggested that this difference rmght be due to &fferent processes or synaptic structures being responsible for the regulation of the two modes of release transmatter Our experiments indicate that there can be large variations m e~ther the frequency of m e p ps or the quantal content of e p ps at a large number of mouse neuromuscular junctions and that the variations are parallel between them These observations confirm results reported by other investigators (Kuno et al, 1971, R a h a m i m o f et al, 1978) Thus, It Is posmble that pre-membrane process(es) may regulate both modes of release of transmatter In common, even though the structural process associated with the plasma membrane &fferentmtes between the two modes (Propst and Ko, 1987) The rate of spontaneous release of transmitter quanta is shown as the frequency of occurrence of m.e p ps The frequency is less dependent on [Ca2+]o than is the quantal content of e p ps (Matthews and Wickelgren, 1977) However, an elevation of [Ca 2 + ], by means of injection of Ca 2+ Ions into nerve terminals (Mde&, 1973; R a h a m l m o f et al, 1978) or by m h i b m o n of matochondnal function (Alnaes and Rahamimoff, 1975, R a h a m l m o f f and Alnaes, 1973) causes a major increase in the frequency of m e p ps Thus, it is clear that the frequency ofm.e.p.ps reflects the availability of [Ca2+], at the resting state, as suggested by many investigators The many fluctuatmns in this rate suggest the probable &vermty of nerve terminals in terms of their abihty to maintain a particular [Ca:+]~.
el
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The evoked release of transmitter quanta depends on [Ca2+]o (Katz, 1969) Internal Ca 2÷ ions present at rest act in concert with Ca 2+ ions that have newly entered the terminal after a nerve impulse (Katz, 1969) This summation of Ca 2+ ions leads to elevation of [Ca 2+]1 and a larger quantal content of e p ps (Alnaes and Rahamlmoff, 1975) Thus, the evoked release probably reflects not only the permeablhty of the nerve-terminal membrane to Ca 2+ ions but also the resting availability of residual Ca 2+ ions inside the terminal This possibility is supported by previous (Grinnel and Pawson, 1989, Rahamlmoff, 1976, R a h a m l m o f f e t al, 1978)results and ours, which showed that the two modes of transmitter release were correlated with each other These results lead to a hypothesis that the diversity of releasablllty at premembrane process(es) is c o m m o n to both modes of release of transmitter quanta If a series of two, closely spaced, nerve impulses are given to the nerve trunk, the second response is larger than the first (Wernlg, 1972, Zucker, 1973) This phenomenon is called as temporal faclhtatlon (Del Castillo and Katz, 1954) The faclhtaUon can be quantltated as the ratio of m2 to m 1 (Del Castlllo and Katz, 1954). This ratio grows as the interval between the twin shocks is reduced (Mallart and Martin, 1968) This phenomenon has been interpreted m terms of the "remdual calcmm" hypothesis (Katz and Mde&, 1968, Rahamlmoff, 1968) It reflects the residual availability of internal Ca 2 + ions, the level which is elevated by the first nerve impulse The residual internal Ca 2 + act with newly available Ca 2+ ions, introduced by the second impulse, leading to an increased rate of transmitter release The ratm of m2 and m 1 is dependent on the quantal content of the first e p p (ml), the higher is the value o f m l the smaller is the ratio of m 2 to m I (Mallart and Martin, 1968) This result held true in the present experiments Furthermore, such a relationship was also observed between the ratio of m2 and rn 1 and the frequency of m e p ps, which probably reflected the residual availability of internal Ca 2+ ions Thus, heterogeneity was also noted in the &stnbutlon of the ratio of m2 to m l However, no parallelism was demonstrated between the ratio of m 2 to m 1 and m 1 or the frequency of m e p ps, perhaps due to the fact that the increase in the ratio of m2 to m l Is an excitatory phenomenon on the mllhsecond scale The first e p p is evoked separately at several seconds and the m e p p reflects the resting condition In summary, even if the structural step assocmted with the membrane of the nerve terminal &fferentlates between spontaneous and evoked release of transmitter quanta (Propst and Ko, 1987), pre-membrane process(es) may regulate both modes of release of transmitter in c o m m o n Thin pre-membrane process(es) does not seem to include the mechamsm for sequestering internal Ca 2+ Ions, which Is suggested to functmn in the temporal facdltatlon of the release of transmitter quanta REFERENCES Alnaes E. and Rahamlmoff R (1975) On the role of mttochondna in transmitter release from motor nerve terminals J. Physiol 248, 285-306
Diversity among motor nerve terminals del Castlllo J and Katz B (1954) Statistical factors Involved m neuromuscular facilitation and depression J Physwl 124, 574-585 Crawford A C (1974) The dependence of evoked transtarter release on external calcium tons at very low mean quantal contents J Phystol 240, 255-278 Erulkar S D and Rahamlmoff R (1976) Peno&c fluctuations m transmitter release at the frog neuromuscular junction J Physwl 256, 20-21P Gnnnell A D and Herrera A A (1980) Physiological regulation of synaptm effectiveness at frog neuromuscular junction J Physwl 307, 301-317 Gnnnell A D and Pawson P A (1989) Dependence of spontaneous release at frog junctions on synaptm strength, external calcium and terminal length J Physwl 418, 397-410 Katz B (1969) The Release of Neural Transmztter Substances pp 1-55 Charles C Thomas, Springfield, Illinois Katz B and Mlledl R (1968) The role of calcmm in neuromuscular faclhtaUon J Physwl 195, 481-492 Kuno M , Turkams S A and Weakly J N (1971) Correlation between nerve terminal size and transmitter release at the neuromuscular junction of the frog J Phystol 213, 545-556 MaUart A and Martin A R (1968) The relation between quantal content and facilitation at the neuromuscular junction of the frog J Physwl 195, 593-604 Matthews G and Wlckelgren W O (1977) On the effect of calcium on frequency of miniature end plate potentials at the frog neuromuscular junction J Physwl 226, 91-101
169
Mde& R (1973) Transmitter release reduced by rejection of calcmm ions into nerve terminals Proc. Roy Soc, B 183, 421-425 Propst J W and Ko C P (1987) Correlation between acuve zone ultrastructure and synaptlc function studied with freeze-fracture of physiologically identified neuromuscular Junctions J, Neuroscz 7, 3654-3664 Rahamlmoff R (1968) A dual effect of calcium ions on neuromuscular faclhtatlon J Physwl 195, 471--480 Rahamlmoff R (1976) The role of calcium m transmRter release at the neuromuscular junction In Motor Innervatzon of Muscle (E&ted by Thesleff S), pp 117-149 Academic Press, New York Rahamlmoff R and Alnaes E (1973) Inhibitory action of ruthenium red on neuromuscular transmission Proc natn Acad Sct U S A 70, 3613-1616 Rahamlmoff R , Erulkar S D , Lev-Tov A and Mere H (1978) Intracellular and extracellular calcmm Ions m transmitter release at the neuromuscular synapse Ann New York Acad Sct 583-598 Rahamlmoff R , Mere H , Erulkar S D and Barenholtx Y (1978) Changes in transmitter release induced by ton containing hposomes Proc natn Acad Scl U S A 75, 5214-5216 Wernlg A (1972) Changes in statistical parameters dunng facthtatlon at the crayfish neuromuscular junction J Physwl 226, 751-759 Zucker R S (1973) Changes In the statistics of transmitter release dunng facthtatton J Physwl 229, 787-810
