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Acetylcholine release from motor-nerve endings in rats treated with neostigmine
EUROPEAN JOURNAL OF PHARMACOLOGY 6 (1969) 281-285. NORTH-HOLLAND PUBLISHING COMP., AMSTERDAM
ACETYLCHOLINE
RELEASE FROM MOTOR-NERVE
RATS TREATED
ENDINGS IN
WITH NEOSTIGMINE
D.V.ROBERTS* and S.THESLEFF Department of Pharmacology, University of Lund, Sweden
Received 20 November 1968
Accepted 24 January 1969
D.V.ROBERTS and S.THESLEFF, Acetylcholine release from motor-nerve endings in rats treated with neostigraine, Euroeapn J. Pharrnacol. 6 (1969) 281-285. Rats injected subcutaneously with 100 t~g neostigmine twice daily for 5 -7 days were less active and appeared to have muscular weakness. The mean endplate potential quantum content of treated rats was lower than normal, as w a s the number of quanta readily available for release. It was concluded that treated rats released less acetylcholine per nerve impulse than did normal rats. The f'mdings ate discussed in relation to the treatment of myasthenia gravis. Acetylcholine release Neostigmine Neuromuscular transmission 1. INTRODUCTION
Myasthenia gravis Quantum content
2. METHODS
Neostigmine (neostigmine methylsulphate) was injected subcutaneously (s.c.) in twice daily doses of 100/ag for 5 - 7 days. To reduce the muscarinic effects of this treatment the rats were given 1 mg atropine s.c. half an hour before each neostigmine injection on the first two days of treatment; on subsequent days the muscarinic effects had decreased and atropine was no longer necessary. Neuromuscular transmission was examined before and after treatment in the tail muscles in vivo as described by Roberts and Thesleff (1965) and in vitro in the isolated diaphragm preparation at 2 8 - 2 9 ° C under conditions similar to those described by Liley (1956). Conventional glass microelectrode techniques were used for recording spontaneous miniature endplate potentials (MEPPs). Estimation of mean ACh quantum content of endplate potentials (EPPs) was made by analysis of the variance o f the amplitude of 25 successive EPPs from each fibre at a stimulation frequency of 1 per second, while transmission was blocked by tubocurarine. The depletion of transmitter stores by more frequent stimulation was studied at frequencies of 10 and 20 per second.
All experiments were made on male albino rats of the Wistar strain with body weights of 1 5 0 - 2 0 0 g.
* Present address: The Physiological Laboratory, University o f Liverpool, England.
Patients with myasthenia gravis overtreated with anticholinesterase drugs may develop severe muscular weakness, sometimes referred to as cholinergic crisis. In view of the fact that very little is known about the effects on neuromuscular transmission of chronic administration of anticholinesterase drugs, a study was undertaken in which rats were given neostigmine twice daily for 5 - 7 days. The doses administered subcutaneously were 1 mg per kilo body weight per day which are comparable with the high dosages sometimes used in the treatment of myasthenia gravis. The effect of this treatment was evaluated in vivo in the tail muscles and in vitro in the diaphragm with intracellular electrode recording techniques. The resuits show that this treatment with neostigmine markedly but reversibly reduced the amount of acetylcholine (ACh) released by each nerve impulse.
282
D.V.ROBERTS and S.THESLEFF
Localisation of the recording to the endplate region was judged by the observation of a fast rising phase of the MEPPs and EPPs. In a series of rats similarly treated, plasma cholinesterase (ChE) activity was estimated by the electrometric method of Tammelin (1953) and the ChE activity at the endplates in the extensor digitorum and tail muscles was visualized by the histochemical method of Koelle and Friedenwald (1949). The length of the incubation time needed for the motor endplate to become visible was used as a rough estimate of the relative amount o f active cholinesterase at the endplates.
;'I
2oo.
r-J
5 m
mOO.
z
['] I.~ • ]°-s.q
3. RESULTS During the 5 - 7 days of treatment with neostigmine, rats were less active than untreated animals and appeared to have muscular weakness as judged by their resistance to handling during each injection. During the first 2 - 3 days of treatment, they ate less food than control rats and failed to gain weight. The first injections o f neostigmine resulted in muscular fasciculation lasting 1 - 2 hr but after a few days of treatment, this effect became less prominent. No recordings were made during the periods when fasciculation was present. Since the rats were injected twice a day, records were obtained at times varying from 5 - 1 7 hr after the last dose. Another series of rats were studied 2 4 - 2 8 hr after the last dose of neostigmine and in a third series, 5 - 6 days were allowed to elapse after the end of treatment before recordings were made. Spontaneous MEPPs were recorded in rive at 11 normal junctions and 6 junctions from treated rats, 5 - 1 7 hr after the last injection. There was no obvious difference in either the frequency or the time course of MEPPs from these two groups. There was, however, a difference in the mean amplitude and amplitude distribution of individual MEPPs as is shown by the histogram (fig. 1). The mean ACh output per nerve impulse in tail muscles in vivo, as estimated by analysis o f the variance of EPP amplitude was reduced from 162 quanta in normal to 89 in treated rats. This reduction persisted for at least 28 hr after the last injection but 5 - 6 days after treatment ended there was a corn-
o%
nO
i5
MEPP AMPLITUDE (mY)
Fig. 1. Histograms of MEPP amplitudes from normal muscles (full line) and from neostigmine treated muscles (broken line). The recordings on which the figure is based were made on tail muscles in rive 5-17 hr after the last neostigmine injection in the case of the treated rats; 948 MEPPs from 11 normal fibres, mean resting membrane potential (RMP) = 80 mV, 857 MEPPs from 6 treated fibres, mean RMP = 75 mV. plete recovery in ACh output. In the isolated diaphragm the mean quantum content was 131 in normal and 72 in muscles from rats after 7 days neostigmine treatment (see table 1). The effect of more rapid nerve stimulation on neuromuscular transmission in rive and in vitro was studied at stimulus frequencies o f 10 and 20 per second. Higher frequencies than these were not used because under such conditions a long lasting presynaptic block developed in many fibres o f the neostigmine treated rats. In the normal rats, however, transmission was effective at frequencies up to 100 per second. Fig. 2 shows the mean quantum content o f the first five EPPs in a train at 10 per second in normal and in treated rats and is constructed from measurements made on tail muscles in vivo at 24 junctions from 3 rats in each group. As has been shown already the mean quantum content o f the first EPP was lower in the treated rats but in both groups successive EPPs showed a similar percentage decrease in quantum content. The results obtained at a stimulus frequency o f 20 per second were qualitatively similar.
ACh RELEASE AND NEOSTIGMINE
283
Table 1
Preparation
Details of experiment
No. of rats used
No. of muscle fibres
ACh output per nerve impulse as mean quantum content
Tail muscle (in vivo)
Untreated
6
29
162 -+ 99 (-+ S.D.)
200 vg neostigmine per day for 5 - 7 days
6
36
89 -+ 29
3
23
85 -+ 23
5 - 6 days after last injection
3
25
165 -+ 48
Untreated
3
30
131 +-25
200 ug neostigmine per day for 7 days
3
24
72 + 4
2 4 - 2 8 hr after last injection
Diaphragm (in vitro)
w o u l d , w h e n e x t r a p o l a t e d , i n t e r s e c t t h e abscissa ( E l m qvist a n d Quastel, 1965). T h i s e s t i m a t i o n was carried o u t for the tail m u s c l e in vivo a n d t h e d i a p h r a g m in TA~L MUSCLE iN VIVO
0
~00 •
DIAPHRAGM IN VITRO
0
150 - ~
150-
50 z L~ ba |
|
.
t
•
i
2
3
4
5
EP9 TRAIN NUMBER
Fig. 2. Graph showing the mean quantum content of the f'trst 5 EPPs in a train in normal (o) and in neostigmine treated (•) rats. Records were made at 24 junctions in tail muscles in vivo in each group of rats, at a stimulus frequency of 10 per second. The procedure used to calculate the mean quantum content was first to express the amplitude of the EPPs as percentage of the f'trst EPP at each junction. Then the average percentage for each EPP was found and used to calculate the mean quantum content, assuming that the quantum content of the first EPP was the same as the mean value found at a stimulus frequency of I per second.
It is possible to e s t i m a t e t h e size o f t h e A C h store readily available for release b y n e r v e i m p u l s e s b y plotting th¢ m e a n q u a n t u m c o n t e n t o f each E P P in a t r a i n a~ainst t h e s u m o f t h e q u a n t a p r e v i o u s l y released. T h e size o f this store is given b y t h e p o i n t at w h i c h a line t h r o u g h t h e initial p a r t o f t h e g r a p h
~oo/ "~",,~ ,oo
0 r..)
X3 I-Z
50
• •*
0 0
! " 250
"
50
' O, I i 1 I 500 0 250 500 QUANTA RELEASED
I
750
Fig. 3. Graphs showing the method of calculation of the maximal number of quanta readily available for release. Mean quantum contents of the f'ttst 5 EPPs in a train were calculated as described in the legend to fig. 2 and the value for each EPP was then plotted against the number of quanta contained in the preceding EPPs. The broken lines were fitted by eye to the initial points on the graphs and extrapolated to cut the abscissae. The point of intersection is considered equal to the maximum number of quanta readily available for release. (For further details see Elmqvist and Quastel, 1965.) a normal rat, 10 stimuli per second, o normal rat, 20/see, • neostigmine treated rat, 10/sec, • neostigmine treated rat, 20/sec.
284
D.V.ROBERTS and S.THESLEFF
vitro in normal and in treated rats at stimulus fre-
quencies of 10 and 20 per second (fig. 3). The sizes of the readily releasable stores were similar in normal tail muscles and diaphragms: neostigmine treatment reduced both these stores by about 50 percent. Evaluation of the ChE activity in plasma and at motor endplates showed that there was no significant difference between treated and untreated rats. It seems therefore that at the times when the records were made on the treated rats, these enzyme systems had already recovered from the inhibition caused by the last injection of neostigmine. This conclusion is in agreement with the observation of smaller MEPPs of normal duration in treated rats.
4. DISCUSSION In the presence of tubocurarine spontaneous MEPPs are not visible and therefore their mean amplitude cannot be used to measure EPP quantum content (= mean EPP amplitude/mean MEPP amplitude). However, the quantal nature of ACh release makes possible a calculation of quantum content based on the variation in the amplitude of successive EPPs. In general terms, the variability of EPP amplitudes varies with their mean quantum content. The two other factors which affect EPP amplitude variability are the electrical noise present in the recording system and the variability in the amplitude of the MEPPs. The electrical noise level was low in all recordings from both normal and treated rats. A comparison of the amplitude distribution of MEPPs (fig. 1) shows that treated rats had smaller MEPPs than untreated rats, but there was a similar variability of MEPP amplitudes about the mean in the two groups. A correction for this difference, if applied to the calculated quantum content, would produce only a small further reduction in the values obtained in treated rats. In view of the already large difference between treated and untreated rats, this correction has been omitted. For these reasons, the finding of a lower quantum content based on the increased variability of EPP amplitudes in treated rats is considered to be valid. There are a number of possible reasons for the smaller MEPPs in treated rats. The lower mean resting membrane potential in the treated rats would account for about one third of the observed difference
in mean MEPP amplitudes. The remaining two-thirds could be due to a reduced amount of ACh contained in each quantum and/or a reduction in postjunctional sensitivity to ACh. However, no measurements of ACh sensitivity were made and so the contribution made by either or both of these factors cannot be evaluated. It appears from these results that the quantum content of EPPs in the diaphragm in vitro and in the tail muscles in vivo are similar, and that both are affected to the same degree by neostigmine treatment. It is of interest that the low quantum content produced by neostigmine in the diaphragm was not restored to normal by immersion of the preparation in the bathing fluid for periods of 3 - 4 hr. This is in line with the observation that the low quantum content in neostigmine treated rats also persisted in vivo for at least 28 hr after the last injection. The quantum content of an EPP is determined by two factors, namely the number of quanta in the nerve terminal which are readily available for release, and the probability of release of individual quanta from this store. The reduced EPP quantum content observed after neostigmine treatment could therefore be due to a reduced probability of release and/or to a smaller store of ACh quanta readily available for release. The results depicted in fig. 3 indicate that the low EPP quantum content in neostigmine treated rats is due to a reduced store of readily available ACh. This is shown by the parallel shift in the dotted line drawn through the initial points on the graphs. Had the low quantum output been due to a decreased probability of release the slope of the line, but not its point of intersection with the abscissae, would have changed. Little can be said at the moment about the way in which neostigmine, or possibly one of its metabolites, reduces the number of quanta readily available for re, lease from nerve endings. The effect is long lasting but reversible, and appears not to be directly associated with ChE inhibition. The action is clearly presynaptic and so may be related to the repetitive nerve activity described by Masland and Wigton (1940) and the nerve block at high frequencies of stimulation observed in the present study. The effect of neostigmine therapy on neuromuscular transmission in myasthenic patients is complex. The basic defect in myasthenia gravis appears to be a small quantum size which can be compensated, to a
ACh RELEASE AND NEOSTIGMINE limited extent, by inhibition of endplate ChE. The results of this preliminary study of the action of neostigmine given chronically in high dosage indicate that the depression of neuromuscular transmission by excessive use of the drug may be due in part to a low EPP q u a n t u m content. To this must be added the possibilities of a nerve block as observed at high frequency stimulation and a postjunctional blocking action due to neostigmine/cholinergic receptor binding. The dosage of neostigmine to be aimed at in the treatment of myasthenic patients should therefore be one which is sufficient to maintain optimal facilitation of neuromuscular transmission by ChE inhibition but not one so high that the depressant actions of the drug become prominent.
ACKNOWLEDGEMENTS One of us (D.V.Roberts) was in receipt of a travel grant from the Wellcome Trust. We are greatly indebted to Dr. J. Lundin from the Research Institute of National Defence, Dept. 1, Sundbyberg, who made plasma cholinesterase determinations and to Dr. B.Soncsson, Dept. of Anatomy, Uni-
285
versity of Lund, for his studies of cholinesterase activity in motor endplates. The study was supported by a research grant (B69-14X-738-04A) from the Swedish Mecical Research Council, Stockholm, Sweden.
REFERENCES Elmqvist, D. and D.M.J.Quastel, 1965, A quantitative study of end-plate potentials in isolated human muscles J. Physiol. 178, 505. Koelle, G.B. and J.Friedenwald, 1949, A histochemical method for localising cholinesterase activity, Proc. Soc. Exptl. Biol. 70, 617. Liley, A.W., 1956, An investigation of spontaneous activity at the neuromuscular junction of the rat, J. Physiol. 132, 650. Masland, R.L. and R.S.Wigton, 1940, Nerve activity accompanying fasciculation produced by prostigmin, J. Neurophysiol. 3, 269. Roberts, D.V. and S.Thesleff, 1965, Neuromuscular transmission in vivo and the actions of decamethonium: a micro-electrode study, Acta anaesth. Scand. 9, 165. Tammelin, L.-E., 1953, An electromettic method for the determination of cholinesterase activity, Scand. J. Clin. Lab. Invest. 5,267.
ACETYLCHOLINE
RELEASE FROM MOTOR-NERVE
RATS TREATED
ENDINGS IN
WITH NEOSTIGMINE
D.V.ROBERTS* and S.THESLEFF Department of Pharmacology, University of Lund, Sweden
Received 20 November 1968
Accepted 24 January 1969
D.V.ROBERTS and S.THESLEFF, Acetylcholine release from motor-nerve endings in rats treated with neostigraine, Euroeapn J. Pharrnacol. 6 (1969) 281-285. Rats injected subcutaneously with 100 t~g neostigmine twice daily for 5 -7 days were less active and appeared to have muscular weakness. The mean endplate potential quantum content of treated rats was lower than normal, as w a s the number of quanta readily available for release. It was concluded that treated rats released less acetylcholine per nerve impulse than did normal rats. The f'mdings ate discussed in relation to the treatment of myasthenia gravis. Acetylcholine release Neostigmine Neuromuscular transmission 1. INTRODUCTION
Myasthenia gravis Quantum content
2. METHODS
Neostigmine (neostigmine methylsulphate) was injected subcutaneously (s.c.) in twice daily doses of 100/ag for 5 - 7 days. To reduce the muscarinic effects of this treatment the rats were given 1 mg atropine s.c. half an hour before each neostigmine injection on the first two days of treatment; on subsequent days the muscarinic effects had decreased and atropine was no longer necessary. Neuromuscular transmission was examined before and after treatment in the tail muscles in vivo as described by Roberts and Thesleff (1965) and in vitro in the isolated diaphragm preparation at 2 8 - 2 9 ° C under conditions similar to those described by Liley (1956). Conventional glass microelectrode techniques were used for recording spontaneous miniature endplate potentials (MEPPs). Estimation of mean ACh quantum content of endplate potentials (EPPs) was made by analysis of the variance o f the amplitude of 25 successive EPPs from each fibre at a stimulation frequency of 1 per second, while transmission was blocked by tubocurarine. The depletion of transmitter stores by more frequent stimulation was studied at frequencies of 10 and 20 per second.
All experiments were made on male albino rats of the Wistar strain with body weights of 1 5 0 - 2 0 0 g.
* Present address: The Physiological Laboratory, University o f Liverpool, England.
Patients with myasthenia gravis overtreated with anticholinesterase drugs may develop severe muscular weakness, sometimes referred to as cholinergic crisis. In view of the fact that very little is known about the effects on neuromuscular transmission of chronic administration of anticholinesterase drugs, a study was undertaken in which rats were given neostigmine twice daily for 5 - 7 days. The doses administered subcutaneously were 1 mg per kilo body weight per day which are comparable with the high dosages sometimes used in the treatment of myasthenia gravis. The effect of this treatment was evaluated in vivo in the tail muscles and in vitro in the diaphragm with intracellular electrode recording techniques. The resuits show that this treatment with neostigmine markedly but reversibly reduced the amount of acetylcholine (ACh) released by each nerve impulse.
282
D.V.ROBERTS and S.THESLEFF
Localisation of the recording to the endplate region was judged by the observation of a fast rising phase of the MEPPs and EPPs. In a series of rats similarly treated, plasma cholinesterase (ChE) activity was estimated by the electrometric method of Tammelin (1953) and the ChE activity at the endplates in the extensor digitorum and tail muscles was visualized by the histochemical method of Koelle and Friedenwald (1949). The length of the incubation time needed for the motor endplate to become visible was used as a rough estimate of the relative amount o f active cholinesterase at the endplates.
;'I
2oo.
r-J
5 m
mOO.
z
['] I.~ • ]°-s.q
3. RESULTS During the 5 - 7 days of treatment with neostigmine, rats were less active than untreated animals and appeared to have muscular weakness as judged by their resistance to handling during each injection. During the first 2 - 3 days of treatment, they ate less food than control rats and failed to gain weight. The first injections o f neostigmine resulted in muscular fasciculation lasting 1 - 2 hr but after a few days of treatment, this effect became less prominent. No recordings were made during the periods when fasciculation was present. Since the rats were injected twice a day, records were obtained at times varying from 5 - 1 7 hr after the last dose. Another series of rats were studied 2 4 - 2 8 hr after the last dose of neostigmine and in a third series, 5 - 6 days were allowed to elapse after the end of treatment before recordings were made. Spontaneous MEPPs were recorded in rive at 11 normal junctions and 6 junctions from treated rats, 5 - 1 7 hr after the last injection. There was no obvious difference in either the frequency or the time course of MEPPs from these two groups. There was, however, a difference in the mean amplitude and amplitude distribution of individual MEPPs as is shown by the histogram (fig. 1). The mean ACh output per nerve impulse in tail muscles in vivo, as estimated by analysis o f the variance of EPP amplitude was reduced from 162 quanta in normal to 89 in treated rats. This reduction persisted for at least 28 hr after the last injection but 5 - 6 days after treatment ended there was a corn-
o%
nO
i5
MEPP AMPLITUDE (mY)
Fig. 1. Histograms of MEPP amplitudes from normal muscles (full line) and from neostigmine treated muscles (broken line). The recordings on which the figure is based were made on tail muscles in rive 5-17 hr after the last neostigmine injection in the case of the treated rats; 948 MEPPs from 11 normal fibres, mean resting membrane potential (RMP) = 80 mV, 857 MEPPs from 6 treated fibres, mean RMP = 75 mV. plete recovery in ACh output. In the isolated diaphragm the mean quantum content was 131 in normal and 72 in muscles from rats after 7 days neostigmine treatment (see table 1). The effect of more rapid nerve stimulation on neuromuscular transmission in rive and in vitro was studied at stimulus frequencies o f 10 and 20 per second. Higher frequencies than these were not used because under such conditions a long lasting presynaptic block developed in many fibres o f the neostigmine treated rats. In the normal rats, however, transmission was effective at frequencies up to 100 per second. Fig. 2 shows the mean quantum content o f the first five EPPs in a train at 10 per second in normal and in treated rats and is constructed from measurements made on tail muscles in vivo at 24 junctions from 3 rats in each group. As has been shown already the mean quantum content o f the first EPP was lower in the treated rats but in both groups successive EPPs showed a similar percentage decrease in quantum content. The results obtained at a stimulus frequency o f 20 per second were qualitatively similar.
ACh RELEASE AND NEOSTIGMINE
283
Table 1
Preparation
Details of experiment
No. of rats used
No. of muscle fibres
ACh output per nerve impulse as mean quantum content
Tail muscle (in vivo)
Untreated
6
29
162 -+ 99 (-+ S.D.)
200 vg neostigmine per day for 5 - 7 days
6
36
89 -+ 29
3
23
85 -+ 23
5 - 6 days after last injection
3
25
165 -+ 48
Untreated
3
30
131 +-25
200 ug neostigmine per day for 7 days
3
24
72 + 4
2 4 - 2 8 hr after last injection
Diaphragm (in vitro)
w o u l d , w h e n e x t r a p o l a t e d , i n t e r s e c t t h e abscissa ( E l m qvist a n d Quastel, 1965). T h i s e s t i m a t i o n was carried o u t for the tail m u s c l e in vivo a n d t h e d i a p h r a g m in TA~L MUSCLE iN VIVO
0
~00 •
DIAPHRAGM IN VITRO
0
150 - ~
150-
50 z L~ ba |
|
.
t
•
i
2
3
4
5
EP9 TRAIN NUMBER
Fig. 2. Graph showing the mean quantum content of the f'trst 5 EPPs in a train in normal (o) and in neostigmine treated (•) rats. Records were made at 24 junctions in tail muscles in vivo in each group of rats, at a stimulus frequency of 10 per second. The procedure used to calculate the mean quantum content was first to express the amplitude of the EPPs as percentage of the f'trst EPP at each junction. Then the average percentage for each EPP was found and used to calculate the mean quantum content, assuming that the quantum content of the first EPP was the same as the mean value found at a stimulus frequency of I per second.
It is possible to e s t i m a t e t h e size o f t h e A C h store readily available for release b y n e r v e i m p u l s e s b y plotting th¢ m e a n q u a n t u m c o n t e n t o f each E P P in a t r a i n a~ainst t h e s u m o f t h e q u a n t a p r e v i o u s l y released. T h e size o f this store is given b y t h e p o i n t at w h i c h a line t h r o u g h t h e initial p a r t o f t h e g r a p h
~oo/ "~",,~ ,oo
0 r..)
X3 I-Z
50
• •*
0 0
! " 250
"
50
' O, I i 1 I 500 0 250 500 QUANTA RELEASED
I
750
Fig. 3. Graphs showing the method of calculation of the maximal number of quanta readily available for release. Mean quantum contents of the f'ttst 5 EPPs in a train were calculated as described in the legend to fig. 2 and the value for each EPP was then plotted against the number of quanta contained in the preceding EPPs. The broken lines were fitted by eye to the initial points on the graphs and extrapolated to cut the abscissae. The point of intersection is considered equal to the maximum number of quanta readily available for release. (For further details see Elmqvist and Quastel, 1965.) a normal rat, 10 stimuli per second, o normal rat, 20/see, • neostigmine treated rat, 10/sec, • neostigmine treated rat, 20/sec.
284
D.V.ROBERTS and S.THESLEFF
vitro in normal and in treated rats at stimulus fre-
quencies of 10 and 20 per second (fig. 3). The sizes of the readily releasable stores were similar in normal tail muscles and diaphragms: neostigmine treatment reduced both these stores by about 50 percent. Evaluation of the ChE activity in plasma and at motor endplates showed that there was no significant difference between treated and untreated rats. It seems therefore that at the times when the records were made on the treated rats, these enzyme systems had already recovered from the inhibition caused by the last injection of neostigmine. This conclusion is in agreement with the observation of smaller MEPPs of normal duration in treated rats.
4. DISCUSSION In the presence of tubocurarine spontaneous MEPPs are not visible and therefore their mean amplitude cannot be used to measure EPP quantum content (= mean EPP amplitude/mean MEPP amplitude). However, the quantal nature of ACh release makes possible a calculation of quantum content based on the variation in the amplitude of successive EPPs. In general terms, the variability of EPP amplitudes varies with their mean quantum content. The two other factors which affect EPP amplitude variability are the electrical noise present in the recording system and the variability in the amplitude of the MEPPs. The electrical noise level was low in all recordings from both normal and treated rats. A comparison of the amplitude distribution of MEPPs (fig. 1) shows that treated rats had smaller MEPPs than untreated rats, but there was a similar variability of MEPP amplitudes about the mean in the two groups. A correction for this difference, if applied to the calculated quantum content, would produce only a small further reduction in the values obtained in treated rats. In view of the already large difference between treated and untreated rats, this correction has been omitted. For these reasons, the finding of a lower quantum content based on the increased variability of EPP amplitudes in treated rats is considered to be valid. There are a number of possible reasons for the smaller MEPPs in treated rats. The lower mean resting membrane potential in the treated rats would account for about one third of the observed difference
in mean MEPP amplitudes. The remaining two-thirds could be due to a reduced amount of ACh contained in each quantum and/or a reduction in postjunctional sensitivity to ACh. However, no measurements of ACh sensitivity were made and so the contribution made by either or both of these factors cannot be evaluated. It appears from these results that the quantum content of EPPs in the diaphragm in vitro and in the tail muscles in vivo are similar, and that both are affected to the same degree by neostigmine treatment. It is of interest that the low quantum content produced by neostigmine in the diaphragm was not restored to normal by immersion of the preparation in the bathing fluid for periods of 3 - 4 hr. This is in line with the observation that the low quantum content in neostigmine treated rats also persisted in vivo for at least 28 hr after the last injection. The quantum content of an EPP is determined by two factors, namely the number of quanta in the nerve terminal which are readily available for release, and the probability of release of individual quanta from this store. The reduced EPP quantum content observed after neostigmine treatment could therefore be due to a reduced probability of release and/or to a smaller store of ACh quanta readily available for release. The results depicted in fig. 3 indicate that the low EPP quantum content in neostigmine treated rats is due to a reduced store of readily available ACh. This is shown by the parallel shift in the dotted line drawn through the initial points on the graphs. Had the low quantum output been due to a decreased probability of release the slope of the line, but not its point of intersection with the abscissae, would have changed. Little can be said at the moment about the way in which neostigmine, or possibly one of its metabolites, reduces the number of quanta readily available for re, lease from nerve endings. The effect is long lasting but reversible, and appears not to be directly associated with ChE inhibition. The action is clearly presynaptic and so may be related to the repetitive nerve activity described by Masland and Wigton (1940) and the nerve block at high frequencies of stimulation observed in the present study. The effect of neostigmine therapy on neuromuscular transmission in myasthenic patients is complex. The basic defect in myasthenia gravis appears to be a small quantum size which can be compensated, to a
ACh RELEASE AND NEOSTIGMINE limited extent, by inhibition of endplate ChE. The results of this preliminary study of the action of neostigmine given chronically in high dosage indicate that the depression of neuromuscular transmission by excessive use of the drug may be due in part to a low EPP q u a n t u m content. To this must be added the possibilities of a nerve block as observed at high frequency stimulation and a postjunctional blocking action due to neostigmine/cholinergic receptor binding. The dosage of neostigmine to be aimed at in the treatment of myasthenic patients should therefore be one which is sufficient to maintain optimal facilitation of neuromuscular transmission by ChE inhibition but not one so high that the depressant actions of the drug become prominent.
ACKNOWLEDGEMENTS One of us (D.V.Roberts) was in receipt of a travel grant from the Wellcome Trust. We are greatly indebted to Dr. J. Lundin from the Research Institute of National Defence, Dept. 1, Sundbyberg, who made plasma cholinesterase determinations and to Dr. B.Soncsson, Dept. of Anatomy, Uni-
285
versity of Lund, for his studies of cholinesterase activity in motor endplates. The study was supported by a research grant (B69-14X-738-04A) from the Swedish Mecical Research Council, Stockholm, Sweden.
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