Br.J. Anaesth. (1977), 49, 217
THE EFFECT OF AGE ON THE SAFETY FACTOR IN NEUROMUSCULAR TRANSMISSION IN THE ISOLATED DIAPHRAGM OF THE RAT S. S. KELLY AND D. V. ROBERTS SUMMARY
It is still not known if infants are more sensitive than adults to non-depolarizing muscle relaxants. Goudsouzian and colleagues (1975) have recently summarized the conflicting evidence on this topic and it appears that infants are found to be more sensitive when the assessment is made on the basis of respiratory function. When direct measurements of neuromuscular function are made, as for example by electromyography or twitch tension recording, there is no difference in sensitivity. These authors suggested that the apparent greater sensitivity of infants may reflect a difference in some aspects of pulmonary function rather than in neuromuscular sensitivity to non-depolarizing relaxants. This question may be resolved by direct investigation of neuromuscular transmission in the muscles of respiration. This study presents evidence, obtained from rat diaphragms in vitro, to show how the safety factor of neuromuscular transmission changes with age. Neuromuscular transmission depends on four main factors : (1) The quantity of acetylcholine released by each motor nerve impulse. (2) The sensitivity of the post-junctional cholinoceptors to acetylcholine. (3) The excitation threshold of the muscle fibre S. S. KELLY, PH.D.; D. V. ROBERTS, B.SC, M.D.J The
membrane adjacent to the end-plate region. (4) The level of activity of the acetylcholinesterase present at the junction. The first factor may be subdivided into the number of acetylcholine quanta released by each nerve impulse, and the amount of acetylcholine contained in each quantum. It is possible to calculate the number of quanta released by an analysis of the variance in the amplitude of end-plate potentials. Measurement of the amplitude of spontaneous miniature end-plate potentials (MEPPs) provides information about the depolarizing action of a single quantum on the post-junctional membrane, but it does not differentiate between changes in acetylcholine content per quantum and changes in postjunctional sensitivity to acetylcholine. Nevertheless, by comparing the product of EPP quantal content and post-junctional depolarization per quantum, with the excitation threshold (factor 3), it is possible to arrive at a value for the safety factor in neuromuscular transmission. Under normal circumstances, motornerve endings release more acetylcholine than is required to ensure action potential formation in skeletal muscle and this is termed the safety factor. Non-depolarizing relaxants have to overcome this safety factor before electromyographic or twitch tension evidence of neuromuscular failure develops. It follows that a reduction in safety factor will lead to an increased sensitivity to non-depolarizing relaxants.
Physiological Laboratory, University of Liverpool, P.O. Box 147, Liverpool L69 3BX. Correspondence to D. V. R.
The fourth factor, the level of neuromuscular cholinesterase activity, has not been considered. If
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An analysis of neuromuscular transmission has been made in phrenic nerve/diaphragm preparations from male rats aged 30 days or 110 days. The amplitude of miniature end-plate potentials was found to decrease with age, being 0.969 ± SEM 0.058 mV at 30 days and 0.510 ± SEM 0.031 mV at 110 days. Over the same period, the quantum content of the first end-plate potential of a train of 40 at 10 Hz, increased from 144.5, SEM+11.1, -10.4 to 346, SEM + 41.4, -37.0. A corresponding change was observed also in the average quantum contents of the last 30 end-plate potentials of each train; from 50.6, SEM + 3.5, - 3 . 2 , to 138.9, SEM+15.0, -13.6. The safety factor for neuromuscular transmission, calculated from these measured parameters, was found at 30 days to be only 70-80% of that at 110 days. It was estimated that the lower safety factor found in young rats was approximately equivalent to the neuromuscular blocking action of a dose of, at least, 0.0225 mg/kg of d-tubocurarine. Extrapolation of these results to man would support previous reports of increased sensitivity to d-tubocurarine in neonates.
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age-related changes occur in this parameter, it is not likely that they would invalidate the results of this investigation. METHODS
(2) mean EPP quantum content (mean EPP amplitude)2 variance of EPP amplitude Because all quanta from any one nerve ending do not produce the same depolarization, a correction must be applied to EPP variance to take account of the variance of quantum depolarization about the mean. A further correction is required to remove the small increase in EPP variance resulting from background noise of the recording system. Both corrections have been applied to the EPP variances used in this
_ MEPP amplitude (mV) x EPP quantum content ~ RMP-excitation threshold (mV) The safety factor therefore indicates the number of times the amount of released acetylcholine exceeds that which is just sufficient to ensure action potential formation. When the safety factor is less than one, transmission from nerve to muscle is incomplete. There was no difference in resting membrane potential between the two age groups and the average RMP of all muscle fibres investigated in this study was 70.85 ± 0.96 (mean + 1SEM) mV. The excitation threshold was taken as —56 mV, this being the mean of measured thresholds in several muscle fibres. This agrees closely with the value of 53.45 ± 0.39 (SEM) mV obtained by Marshall and Ward (1974). Because the end-plate membrane response to increases in EPP quantum content is non-linear, a correction must be applied to the voltage parameters of the equation for the safety factor. In practice, it was easier and produced the same end result to make a single correction to the depolarization required to initiate a muscle action potential. Accordingly, the 15-mV difference between the resting membrane potential and the excitation threshold has been increased to 20.5 mV in the manner described by Martin (1955). All results are given as mean ± 1 SEM, with n — number of observations contributing to each value. MEPP amplitudes are distributed normally and, for this parameter, arithmetic means are used. The quantum contents of first and plateau EPPs are distributed log-normally, hence the use of geometric means and the different values for SEM. The standard error of the mean for the values for safety factor was calculated from the coefficients of variation of MEPP amplitude and EPP quantum content, using the formula
cv\v = cv*x.cv\+cv\+cv*v where CV = coefficient of variation (Colquhoun, 1971).
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Male C.F.H.B. rats in age groups of 30 days and 110 days were used. Phrenic-nerve/diaphragm preparations were made under ether anaesthesia and used for microelectrode analysis of neuromuscular transmission at 32 °C under conditions similar to those described by Liley (1956). Recordings were made of the spontaneous activity (MEPPs) without tubocurarine, and end-plate potentials (EPPs) obtained in response to 4-s trains of electrical stimuli at a frequency of 10 Hz delivered to the phrenic nerve, in the presence of sufficient tubocurarine to abolish muscle action potentials and prevent twitching. Measurement and calculation of mean MEPP amplitude provided a measure of the depolarization produced by each quantum of acetylcholine. The variance in amplitude of the last 30 EPPs in each train of 40 was calculated and used to provide estimates of the average quantum content of these EPPs, and the quantum content of the first EPP of the train. The validity of the calculation of average quantum content from the variance of EPP amplitude depends on three factors: (1) The quantum contents of successive EPPs conform to a Poisson distribution. (2) A property of the Poisson distribution is that the variance is equal to the mean. (3) The mean quantum content is equal to the mean EPP amplitude (mV) divided by the depolarization produced by each quantum. The following relationships may be derived: (1) quantum depolarization _ variance of EPP amplitude mean EPP amplitude
study. A complete mathematical description of the variance method of measuring quantum content is given by Hubbard, Llinas and Quastel (1969). The size of the safety factor depends on the depolarization produced by each quantum of acetylcholine, the number of quanta released by each nerve impulse and the difference between the resting membrane potential (RMP) and the excitation threshold potential of muscle fibres. Thus: Safety factor
AGE AND NEUROMUSCULAR TRANSMISSION SAFETY FACTOR Statistical comparison between the neuromuscular parameters of 30-day and 110-day-old rats was made using the t test. RESULTS
TABLE I. Effect of age on MEPP amplitude and EPP quantum content {mean ± SEAT), n = number of muscle fibres examined in each group of three rats 30 Day MEPP amplitude (arith. mean) (mV) 1st EPP quantum content (geom. mean) (quanta) Plateau EPP quantum content (geom. mean) (quanta)
110 Day
0.969 + 0.058 0.510 ±0.031 n = 22 n = 33 (JP< 0.001) 346 '4 J40 -10.4 n = 28 n = 30 (P< 0.001)
1144 4 4 >5 5
50.6+U
138.9+gJ
n = 37 n = 30 (P< 0.001)
Using the mean values for MEPP amplitude and EPP quantum content as set out in table I, the safety factor for neuromuscular transmission was calculated (table II). For the first EPP, 30-day-old rats had a safety factor only 80% of that of the 110-day-old rats (0.1>P>0.05). The plateau EPP safety factor of the younger rats was 70% of that of the older animals (P< 0.001). When evaluating these results it should be noted that the standard errors represent the maximum, being derived from the product of the coefficient of variation of the parameters, MEPP
amplitude and EPP quantum content. The calculation takes into account the extreme combinations of high and low MEPP amplitudes and EPP quantum contents. At present there no evidence of a direct relation between MEPP amplitude and EPP quantum TABLE II. Effect of age on safety factor of neuromuscular transmission {mean ±SEM), amp_
+n quantum content" 1
30 Day 1st EPP
6.83
+ 0.81
110 Day 8.61 +0.91
n = 49 n = 62 (0.10>P>0.05 a one-tailed) Plateau EPP
-0.19 58 n = 62 (P< 0.001)
content in individual muscle fibres, and it may be that the true standard errors of the means of the safety factors are less than those given in table II, thereby increasing the significance of the differences between the two age groups. DISCUSSION
The mechanisms responsible for the changes in these parameters of neuromuscular transmission have not been investigated but are probably part of the normal growth process. Muscle fibre diameter is one major factor governing the amplitude of MEPPs (Katz and Thesleff, 1957) and it is, therefore, to be expected that MEPP amplitude will diminish as the rat ages and as its muscle fibre diameter increases. A similar argument may be used to explain the change in quantum output with age. The number of quanta released by each nerve impulse is a function of the number of quanta available for release from the nerve ending and the proportion of this population released by one nerve impulse. It may be supposed that as motor nerve fibres grow in size, the number of quanta stored in the terminals increases also, and thus the number of quanta released by each nerve impulse. The probability of release, calculated from the rundown of EPP amplitude and quantum content at the start of a train, does not change with age over the period 30-110 days (Kelly, 1976). If extrapolation from rat experiments to the human is valid, our results suggest that infants will have a lower safety factor than adults and will therefore be more sensitive to non-depolarizing relaxants.
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The results obtained may be summarized as follows (table I): (1) The MEPP amplitude in the 110-day-old rats was half that found in the 30-day-old rats. (2) The number of acetylcholine quanta released by the first stimulus of a train of 40 at 10 Hz was more than twice as much in the 110-day-old rats as in the 30-day-old rats. (3) The average quantum content of EPPs number 10-40 in trains at 10 Hz, that is ignoring the decrease in EPP amplitude and quantum content at the start of a train, was three times greater in the 110-day-old rats than in the 30-day-old rats. Because the average quantum content multiplied by the rate of stimulation is equal to the rate at which acetylcholine quanta are made available for release, it may be concluded that this function was also three times greater in the older rats.
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In the 30-day-old rats, the same reduction of quantum size to one-fifth of normal would result in an even greater impairment of neuromuscular transmission. If the depolarization per quantum were reduced by d-tubocurarine, similar changes would occur. For the same concentration of d-tubocurarine at the endplates, neuromuscular transmission is more likely to fail in the 30-day-old rats than in the 110-day-old rats. If the same conditions apply to human neonates as to the 30-day-old rats, then a given test dose of d-tubocurarine would produce more "curare-sensitive" individuals in neonates than in older children, because a greater proportion of the neonates have safety factors nearer to the threshold value of 1. This may explain the observation of Goudsouzian and colleagues (1975) that neonates demonstrated a wider range of sensitivity to d-tubocurarine than did older children. Direct electrophysiological evidence of similar age-related changes in neuromuscular parameters of human skeletal muscle is not available yet, and there are obvious species differences in the degree of maturity at birth. For example, the rat first opens its
eyes 2-3 weeks after birth and there is little muscular activity during this period. Redfern (1970) has shown that this period is one of rapid change in some characteristics of neuromuscular transmission. In particular, he found that the number of quanta released by each nerve impulse was too small to result in depletion of the quantal store of acetylcholine, as indicated by the absence of a decrease in amplitude of the first few EPPs of a train. However, the current investigation demonstrates that neuromuscular transmission in the rat at 30 days has matured to a degree at which quantum output is large enough to deplete this store and produce a run-down of EPP amplitudes. Blackhall and colleagues (1969) observed an electromyographic run-down in a 1-day-old infant and Goudsouzian and colleagues (1975) have used the run-down in the myogram produced by trains of four stimuli at 2 Hz to monitor neuromuscular blockade in infants less than 10 days old. For these reasons, it is concluded that the 30-day-old rat and the human neonate are qualitatively similar in respect of neuromuscular transmission, although there may be quantitative differences in MEPP amplitude and EPP quantum content. In evaluating the results of studies such as that of Goudsouzian and colleagues (1975) the safety factor should be considered. In their investigations the maximum depression of muscle twitch amplitude was noted after each successive injection of d-tubocurarine. A dose-response relation was obtained by plotting corresponding values of dose and depression of twitch on log-probability paper. However, because twitch amplitude is reduced only after the safety factor has been overcome by d-tubocurarine, this procedure underestimates the neuromuscular blocking action of the first dose of the drug. The error produced in the dose-response relation will vary with the size of the safety factor. The size of the safety factor will influence also the latency between the injection of each dose of the drug and the first sign of depressed muscle activity, an effect which may be seen in figure 1 of the communication by Goudsouzian and colleagues (1975). These differences account for the positive results of the present study and the negative conclusions of Goudsouzian and colleagues (1975). The partial loss of safety factor may be regarded as a degree of neuromuscular blockade, insufficient to result in any overt sign of weakness in a recording of twitch tension. It resembles the effect of a small dose of d-tubocurarine which is insufficient to produce any neuromuscular blockade but which renders the
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The two parameters of neuromuscular transmission, quantum size and EPP quantum content, change reciprocally with age (table I). Thus at 30 days a greater quantum size effectively offsets the smaller EPP quantum content, while at 110 days the opposite relationship exists. Deviation from this normal pattern of maturation may produce a reduction in safety factor and the possibility of neuromuscular failure. For example, the characteristic weakness of myasthenia gravis results from a reduction in depolarization produced by each quantum of acetylcholine, and not from a reduction in the EPP quantum content. From microelectrode recording from motor end-plates in human intercostal muscle it has been shown that the mean MEPP amplitude in myasthenic patients was one-fifth of that found in non-myasthenic subjects (Elmqvist et al., 1964). If the same reduction in quantum size were to occur in the 110-day-old rats of this study, the safety factor for the first EPP would be 2.07, while that for the plateau EPP's would be only 0.81. Under such conditions, the muscle fibres of the diaphragm would respond, on average, to only the first and perhaps the second nerve impulses of each train. For succeeding impulses, the EPP amplitude would be below threshold and neuromuscular transmission would fail. In vivo, this sequence of events would curtail the inspiratory action of the diaphragm and so reduce pulmonary ventilation.
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AGE AND NEUROMUSCULAR TRANSMISSION SAFETY FACTOR
in the concentration required to abolish twitching. Small differences in safety factor, such as those between 30- and 110-day-old rats, would not be deteaed in the larger reduoion. This comment may be applied to the study of Goudsouzian and colleagues (1975) and demonstrates the sensitivity of the quantitative methods of measurement of neuromuscular transmission utilized in the present investigation. ACKNOWLEDGEMENT
This study was carried out while S. S. Kelly was an M.R.C. Research Student. REFERENCES
Blackhall, M. I., Buckley, G. A., Roberts, D. V., Roberts, J. B., Thomas, B. H., and Wilson, A. (1969). Druginduced neonatal myasthenia. J. Obstet. Gynaecol. Br. Commonw., 76, 157. Colquhoun, D. (1971). Lectures on Biostatistics. Oxford: Clarendon. Elmqvist, D., Hofmann, W. W., Kugelberg, J., and Quastel, D. M. J. (1964). An electrophysiological investigation of neuromuscular transmission in myasthenia gravis. J. Physiol. {Lond.), 174, 417. Fatt, P., and Katz, B. (1952). Spontaneous subthreshold activity at motor nerve endings. J. Physiol. {Lond.), 117, 109. Goudsouzian, N. G., Donlon, J. V., Savarese, J. J., and Ryan, J. F. (1975). Re-evaluation of dosage and duration of action of d-tubocurarine in the pediatric age group. Anesthesiology, 43, 416. Hubbard, J. I., Llinas, R., and Quastel, D. M. J. (1969). Electrophysiological Analysis of Synoptic Transmission. London: Edward Arnold (Publishers) Ltd. Katz, B., and Thesleff, S. (1957). On the factors which determine the amplitude of the "miniature end-plate potential". J. Physiol. {Lond.), 137, 267. Kelly, S. S. (1976). The effects of age and nutrition on neuromuscular transmission. Ph.D. Thesis, University of Liverpool. Liley, A. W. (1956). An investigation of spontaneous activity at the neuromuscular junction of the rat. J. Physiol. {Lond.), 132, 650. Marshall, M. W., and Ward, M. R. (1974). Anode break excitation in denervated rat skeletal muscle fibres. J. Physiol. {Lond.), 236, 413. Martin, A. R. (1955). A further study of the statistical composition of the end-plate potential. J. Physiol. {Lond.), 130, 114. Redfern, P. A. (1970). Neuromuscular transmission in newborn rats. J. Physiol. {Lond.), 209, 701. L'EFFET DE L'AGE SECURITE DANS NEUROMUSCULAIRE ISOLE
SUR LE FACTEUR DE LA TRANSMISSION SUR LE DIAPHRAGME DU RAT
RESUME
Une analyse de la transmission neuromusculaire a ete faite dans les preparations de nerf phrenique/diaphragme de
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subject more sensitive to a further dose of the drug. In terms of the dose-response curve, a loss of safety factor would be reflected by a shift of the curve to the left and the magnitude of displacement would be equal to the dose of d-tubocurarine which would produce an equivalent reduction in safety factor. It has been shown that d-tubocurarine, in a concentration of 5 x 10~7 w/v, reduced MEPP amplitude and hence, safety faaor, by 50% (Fatt and Katz, 1952). Such a concentration in the plasma could result from a dose of 0.0225 mg/kg given rapidly i.v. into a plasma volume of 45 ml/kg body weight. The concentration of d-tubocurarine at the end-plates would be marginally less, by dilution in the small amount of interstitial fluid between capillary and end-plate. On this basis, for a 50% loss of safety factor there would be a shift in the dose-response curve equivalent to 0.0225 mg/kg of d-tubocurarine. This is of the same order of magnitude as the standard errors of the means of effective doses of d-tubocurarine found by Goudsouzian and colleagues (1975)'and it is unlikely therefore that their method would detect a difference in safety factor similar to that found between 30-day and 110-day-old rats. A displacement of the dose-response curve to the left may provide an explanation for the greater variance in effective doses of d-tubocurarine noted by Goudsouzian and colleagues (1975). The curve is sigmoid in shape and the initial dose of d-tubocurarine 0.1 mg/kg occurs at the lower inflexion of the curve where the response is insensitive to small differences in dose. A displacement to the left would cause the initial dose to lie on a steeper portion of the curve and larger changes in response would occur. This could increase the variance of the lines drawn through the corresponding dose-response values for each subject and lead to a greater range of effective dose values, particularly at the ED 80 and ED 95 levels. In view of the difference in safety factor between 30-day and 110-day-old rats it might be expected that the diaphragms from the young rats would require less d-tubocurarine in the bathing fluid to produce a given degree of neuromuscular block. However, the concentration of d-tubocurarine was varied within the range 10-«+10%w/v (1.15-1.40x 10~6mol/litre) in order to obtain EPPs of optimal size in relation to background noise and facilitate accurate measurements of their amplitudes. There was no obvious difference in the concentration of d-tubocurarine required to produce these conditions in the two groups of rats. This would be anticipated, in view of the large reduction in safety faaor produced by d-tubocurarine
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DER ALTERSEINFLUSS AUF DEN SICHERHEITSFAKTOR IN NEUROMUSKULAREN U B E R T R A G U N G E N IM ISOLIERTEN
RATTENZWERCHFELL ZUSAMMENFASSUNG
Die neuromuskulare iibertragung wurde in Phrenikus/ Zwerchfell-Praparaten aus 30-Tage oder 110-Tage alten Rattenmannchen untersucht. Die Kleinspannungen an den Nervenendplatten wurden als 0,969 ± SFM (Standardfehler des Mittels) 0,058 mV ftir 30 Tage und 0,510 +SFM 0,031 mV fur 110 Tage ermittelt, d.h. ihre Grosse sank mit steigendem Alter ab. Wahrend der gleichen Zeitspanne stieg der Quantengehalt der ersten Nervenendplatten spannung einer 10-Hz Serie von 40 von 144,5 SFM + 11,1 -10,4 auf 346,0 SFM +41,4 -37,0 an. Nach den Beobachtungen anderte sich der Durchschnittsquantengehalt der letzten 30 Nervenendplattenspannungen jeder
Serie dementsprechend: von 50,6 SFM +3,5 —3,2 auf 138,9 SFM +15,0 -13,6. Es wurde festgestellt, dass der aus diesen gemessenen Parametern errechnete Sicherheitsfaktor fur neuromuskulare iibertragung fur 30 Tage nur 70-80% dessen fiir 110 Tage betrug. Es wurde gescha'tzt, dass der in jungen Ratten festgestellte untere Sicherheitsfaktor ungefahr der neuromuskularen Blockierungswirkung einer Mindestdosis von 0,0225 mg/kg des d-Tubocurarins glich. Eine Extrapolation dieser Ergebnisse auf den Menschen wurde fruhere Berichte uber erhohte d-Tubocurarin-Empfindlichkeit in Neugeborenen bekraftigen. EL EFECTO DE LA EDAD SOBRE EL FACTOR DE SEGURIDAD EN LA TRANSMISION NEUROMUSCULAR EN EL DIAFRAGMA AISLADO DE LA RATA SUMARIO
Se ha efectuado un analisis de transmision neuromuscular en preparaciones de nervio frenico diafragma procedentes de ratos de 30 o 110 dias. La amplitud de los potenciales de placas terminales miniatura se hallo que disminula con la edad, siendo 0,969 + SEM (E.T.M.) 0,058 mV a los 30 dias de edad, y 0,510 ±0,031 E.T.M. mV a los 110 dias. Durante el mismo periodo, el contenido quantum del primer potential de placa terminal de una serie de 40 a 10 Hz aument6 desde 144,5 E.T.M. + 11,1,-10,4 hasta 346 E.T.M. + 41,4, — 37,0. Tambito se observ6 un cambio correspondiente en el contenido promedio de quantum de los ultimos 30 potenciales de placa terminal de cada serie; desde 50,6 E.T.M. +3,5, - 3 , 2 hasta 138,9 E.T.M. +15,0, -13,6. El factor de seguridad para la transmisidn neuromuscular, calculado a partir de estos parametros medidos, se hallo que era a los 30 dias solamente el 70-80% del a los 110 dias. Se calculo que el mas bajo factor de seguridad hallado en los ratos jovenes era aproximadamente a la action bloqueadora neuromuscular de una dosis de, por lo menos, 0,0225 mg/kg de d-tubocurarina. La extrapolaci6n de estos resultados al hombre respaldaria anteriores comunicaciones sobre una mayor sensibilidad a la d-tubocurarina en los neonatos.
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rats males Sges de 30 jours ou de 110 jours. On a trouve que l'amplitude des potentiels des plaques d'extr&nite miniaturisees allait en decroissant avec l'^ge, 6tant de 0,969 + SEM (erreur standard des moyennes) 0,058 mV a 30 jours et de 0,510 ± SEM 0,031 mV a 110 jours. Au cours de la meme periode la teneur du quantum du potentiel de la premiere plaque d'extremite d'une serie de 40 a 10 Hz a augmente de 144,5 SEM + 1 1 , 1 , - 10,4 a 346 SEM +41,4, — 37,0. Une variation correspondante a egalement 6te observee dans les teneurs du quantum moyen des derniers 30 potentiels de plaque d'extremite de chaque serie; de 50,6 SEM +3,5, - 3 , 2 a 138,9 SEM +15,0, -13,6. On a trouve que le facteur de securite pour la transmission neuromusculaire, calcule a partir de ces parametres mesures, n'etait a 30 jours que de 70-80% de celui a 110 jours. On a estime que le facteur de securite plus bas, que Ton a trouve sur les jeunes rats, etait a peu pres equivalent a l'action de blocage neuromusculaire d'une dose d'au moins 0,0225 mg/kg de d-tubocurarine. L'extrapolation de ces rdsultats pour rhomme permet d'appuyer les rapports precedents sur la sensibilite accrue des nouveaux-nes a la d-tubocurarine.
THE EFFECT OF AGE ON THE SAFETY FACTOR IN NEUROMUSCULAR TRANSMISSION IN THE ISOLATED DIAPHRAGM OF THE RAT S. S. KELLY AND D. V. ROBERTS SUMMARY
It is still not known if infants are more sensitive than adults to non-depolarizing muscle relaxants. Goudsouzian and colleagues (1975) have recently summarized the conflicting evidence on this topic and it appears that infants are found to be more sensitive when the assessment is made on the basis of respiratory function. When direct measurements of neuromuscular function are made, as for example by electromyography or twitch tension recording, there is no difference in sensitivity. These authors suggested that the apparent greater sensitivity of infants may reflect a difference in some aspects of pulmonary function rather than in neuromuscular sensitivity to non-depolarizing relaxants. This question may be resolved by direct investigation of neuromuscular transmission in the muscles of respiration. This study presents evidence, obtained from rat diaphragms in vitro, to show how the safety factor of neuromuscular transmission changes with age. Neuromuscular transmission depends on four main factors : (1) The quantity of acetylcholine released by each motor nerve impulse. (2) The sensitivity of the post-junctional cholinoceptors to acetylcholine. (3) The excitation threshold of the muscle fibre S. S. KELLY, PH.D.; D. V. ROBERTS, B.SC, M.D.J The
membrane adjacent to the end-plate region. (4) The level of activity of the acetylcholinesterase present at the junction. The first factor may be subdivided into the number of acetylcholine quanta released by each nerve impulse, and the amount of acetylcholine contained in each quantum. It is possible to calculate the number of quanta released by an analysis of the variance in the amplitude of end-plate potentials. Measurement of the amplitude of spontaneous miniature end-plate potentials (MEPPs) provides information about the depolarizing action of a single quantum on the post-junctional membrane, but it does not differentiate between changes in acetylcholine content per quantum and changes in postjunctional sensitivity to acetylcholine. Nevertheless, by comparing the product of EPP quantal content and post-junctional depolarization per quantum, with the excitation threshold (factor 3), it is possible to arrive at a value for the safety factor in neuromuscular transmission. Under normal circumstances, motornerve endings release more acetylcholine than is required to ensure action potential formation in skeletal muscle and this is termed the safety factor. Non-depolarizing relaxants have to overcome this safety factor before electromyographic or twitch tension evidence of neuromuscular failure develops. It follows that a reduction in safety factor will lead to an increased sensitivity to non-depolarizing relaxants.
Physiological Laboratory, University of Liverpool, P.O. Box 147, Liverpool L69 3BX. Correspondence to D. V. R.
The fourth factor, the level of neuromuscular cholinesterase activity, has not been considered. If
Downloaded from http://bja.oxfordjournals.org/ at University of Western Ontario on June 11, 2015
An analysis of neuromuscular transmission has been made in phrenic nerve/diaphragm preparations from male rats aged 30 days or 110 days. The amplitude of miniature end-plate potentials was found to decrease with age, being 0.969 ± SEM 0.058 mV at 30 days and 0.510 ± SEM 0.031 mV at 110 days. Over the same period, the quantum content of the first end-plate potential of a train of 40 at 10 Hz, increased from 144.5, SEM+11.1, -10.4 to 346, SEM + 41.4, -37.0. A corresponding change was observed also in the average quantum contents of the last 30 end-plate potentials of each train; from 50.6, SEM + 3.5, - 3 . 2 , to 138.9, SEM+15.0, -13.6. The safety factor for neuromuscular transmission, calculated from these measured parameters, was found at 30 days to be only 70-80% of that at 110 days. It was estimated that the lower safety factor found in young rats was approximately equivalent to the neuromuscular blocking action of a dose of, at least, 0.0225 mg/kg of d-tubocurarine. Extrapolation of these results to man would support previous reports of increased sensitivity to d-tubocurarine in neonates.
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age-related changes occur in this parameter, it is not likely that they would invalidate the results of this investigation. METHODS
(2) mean EPP quantum content (mean EPP amplitude)2 variance of EPP amplitude Because all quanta from any one nerve ending do not produce the same depolarization, a correction must be applied to EPP variance to take account of the variance of quantum depolarization about the mean. A further correction is required to remove the small increase in EPP variance resulting from background noise of the recording system. Both corrections have been applied to the EPP variances used in this
_ MEPP amplitude (mV) x EPP quantum content ~ RMP-excitation threshold (mV) The safety factor therefore indicates the number of times the amount of released acetylcholine exceeds that which is just sufficient to ensure action potential formation. When the safety factor is less than one, transmission from nerve to muscle is incomplete. There was no difference in resting membrane potential between the two age groups and the average RMP of all muscle fibres investigated in this study was 70.85 ± 0.96 (mean + 1SEM) mV. The excitation threshold was taken as —56 mV, this being the mean of measured thresholds in several muscle fibres. This agrees closely with the value of 53.45 ± 0.39 (SEM) mV obtained by Marshall and Ward (1974). Because the end-plate membrane response to increases in EPP quantum content is non-linear, a correction must be applied to the voltage parameters of the equation for the safety factor. In practice, it was easier and produced the same end result to make a single correction to the depolarization required to initiate a muscle action potential. Accordingly, the 15-mV difference between the resting membrane potential and the excitation threshold has been increased to 20.5 mV in the manner described by Martin (1955). All results are given as mean ± 1 SEM, with n — number of observations contributing to each value. MEPP amplitudes are distributed normally and, for this parameter, arithmetic means are used. The quantum contents of first and plateau EPPs are distributed log-normally, hence the use of geometric means and the different values for SEM. The standard error of the mean for the values for safety factor was calculated from the coefficients of variation of MEPP amplitude and EPP quantum content, using the formula
cv\v = cv*x.cv\+cv\+cv*v where CV = coefficient of variation (Colquhoun, 1971).
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Male C.F.H.B. rats in age groups of 30 days and 110 days were used. Phrenic-nerve/diaphragm preparations were made under ether anaesthesia and used for microelectrode analysis of neuromuscular transmission at 32 °C under conditions similar to those described by Liley (1956). Recordings were made of the spontaneous activity (MEPPs) without tubocurarine, and end-plate potentials (EPPs) obtained in response to 4-s trains of electrical stimuli at a frequency of 10 Hz delivered to the phrenic nerve, in the presence of sufficient tubocurarine to abolish muscle action potentials and prevent twitching. Measurement and calculation of mean MEPP amplitude provided a measure of the depolarization produced by each quantum of acetylcholine. The variance in amplitude of the last 30 EPPs in each train of 40 was calculated and used to provide estimates of the average quantum content of these EPPs, and the quantum content of the first EPP of the train. The validity of the calculation of average quantum content from the variance of EPP amplitude depends on three factors: (1) The quantum contents of successive EPPs conform to a Poisson distribution. (2) A property of the Poisson distribution is that the variance is equal to the mean. (3) The mean quantum content is equal to the mean EPP amplitude (mV) divided by the depolarization produced by each quantum. The following relationships may be derived: (1) quantum depolarization _ variance of EPP amplitude mean EPP amplitude
study. A complete mathematical description of the variance method of measuring quantum content is given by Hubbard, Llinas and Quastel (1969). The size of the safety factor depends on the depolarization produced by each quantum of acetylcholine, the number of quanta released by each nerve impulse and the difference between the resting membrane potential (RMP) and the excitation threshold potential of muscle fibres. Thus: Safety factor
AGE AND NEUROMUSCULAR TRANSMISSION SAFETY FACTOR Statistical comparison between the neuromuscular parameters of 30-day and 110-day-old rats was made using the t test. RESULTS
TABLE I. Effect of age on MEPP amplitude and EPP quantum content {mean ± SEAT), n = number of muscle fibres examined in each group of three rats 30 Day MEPP amplitude (arith. mean) (mV) 1st EPP quantum content (geom. mean) (quanta) Plateau EPP quantum content (geom. mean) (quanta)
110 Day
0.969 + 0.058 0.510 ±0.031 n = 22 n = 33 (JP< 0.001) 346 '4 J40 -10.4 n = 28 n = 30 (P< 0.001)
1144 4 4 >5 5
50.6+U
138.9+gJ
n = 37 n = 30 (P< 0.001)
Using the mean values for MEPP amplitude and EPP quantum content as set out in table I, the safety factor for neuromuscular transmission was calculated (table II). For the first EPP, 30-day-old rats had a safety factor only 80% of that of the 110-day-old rats (0.1>P>0.05). The plateau EPP safety factor of the younger rats was 70% of that of the older animals (P< 0.001). When evaluating these results it should be noted that the standard errors represent the maximum, being derived from the product of the coefficient of variation of the parameters, MEPP
amplitude and EPP quantum content. The calculation takes into account the extreme combinations of high and low MEPP amplitudes and EPP quantum contents. At present there no evidence of a direct relation between MEPP amplitude and EPP quantum TABLE II. Effect of age on safety factor of neuromuscular transmission {mean ±SEM), amp_
+n quantum content" 1
30 Day 1st EPP
6.83
+ 0.81
110 Day 8.61 +0.91
n = 49 n = 62 (0.10>P>0.05 a one-tailed) Plateau EPP
-0.19 58 n = 62 (P< 0.001)
content in individual muscle fibres, and it may be that the true standard errors of the means of the safety factors are less than those given in table II, thereby increasing the significance of the differences between the two age groups. DISCUSSION
The mechanisms responsible for the changes in these parameters of neuromuscular transmission have not been investigated but are probably part of the normal growth process. Muscle fibre diameter is one major factor governing the amplitude of MEPPs (Katz and Thesleff, 1957) and it is, therefore, to be expected that MEPP amplitude will diminish as the rat ages and as its muscle fibre diameter increases. A similar argument may be used to explain the change in quantum output with age. The number of quanta released by each nerve impulse is a function of the number of quanta available for release from the nerve ending and the proportion of this population released by one nerve impulse. It may be supposed that as motor nerve fibres grow in size, the number of quanta stored in the terminals increases also, and thus the number of quanta released by each nerve impulse. The probability of release, calculated from the rundown of EPP amplitude and quantum content at the start of a train, does not change with age over the period 30-110 days (Kelly, 1976). If extrapolation from rat experiments to the human is valid, our results suggest that infants will have a lower safety factor than adults and will therefore be more sensitive to non-depolarizing relaxants.
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The results obtained may be summarized as follows (table I): (1) The MEPP amplitude in the 110-day-old rats was half that found in the 30-day-old rats. (2) The number of acetylcholine quanta released by the first stimulus of a train of 40 at 10 Hz was more than twice as much in the 110-day-old rats as in the 30-day-old rats. (3) The average quantum content of EPPs number 10-40 in trains at 10 Hz, that is ignoring the decrease in EPP amplitude and quantum content at the start of a train, was three times greater in the 110-day-old rats than in the 30-day-old rats. Because the average quantum content multiplied by the rate of stimulation is equal to the rate at which acetylcholine quanta are made available for release, it may be concluded that this function was also three times greater in the older rats.
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In the 30-day-old rats, the same reduction of quantum size to one-fifth of normal would result in an even greater impairment of neuromuscular transmission. If the depolarization per quantum were reduced by d-tubocurarine, similar changes would occur. For the same concentration of d-tubocurarine at the endplates, neuromuscular transmission is more likely to fail in the 30-day-old rats than in the 110-day-old rats. If the same conditions apply to human neonates as to the 30-day-old rats, then a given test dose of d-tubocurarine would produce more "curare-sensitive" individuals in neonates than in older children, because a greater proportion of the neonates have safety factors nearer to the threshold value of 1. This may explain the observation of Goudsouzian and colleagues (1975) that neonates demonstrated a wider range of sensitivity to d-tubocurarine than did older children. Direct electrophysiological evidence of similar age-related changes in neuromuscular parameters of human skeletal muscle is not available yet, and there are obvious species differences in the degree of maturity at birth. For example, the rat first opens its
eyes 2-3 weeks after birth and there is little muscular activity during this period. Redfern (1970) has shown that this period is one of rapid change in some characteristics of neuromuscular transmission. In particular, he found that the number of quanta released by each nerve impulse was too small to result in depletion of the quantal store of acetylcholine, as indicated by the absence of a decrease in amplitude of the first few EPPs of a train. However, the current investigation demonstrates that neuromuscular transmission in the rat at 30 days has matured to a degree at which quantum output is large enough to deplete this store and produce a run-down of EPP amplitudes. Blackhall and colleagues (1969) observed an electromyographic run-down in a 1-day-old infant and Goudsouzian and colleagues (1975) have used the run-down in the myogram produced by trains of four stimuli at 2 Hz to monitor neuromuscular blockade in infants less than 10 days old. For these reasons, it is concluded that the 30-day-old rat and the human neonate are qualitatively similar in respect of neuromuscular transmission, although there may be quantitative differences in MEPP amplitude and EPP quantum content. In evaluating the results of studies such as that of Goudsouzian and colleagues (1975) the safety factor should be considered. In their investigations the maximum depression of muscle twitch amplitude was noted after each successive injection of d-tubocurarine. A dose-response relation was obtained by plotting corresponding values of dose and depression of twitch on log-probability paper. However, because twitch amplitude is reduced only after the safety factor has been overcome by d-tubocurarine, this procedure underestimates the neuromuscular blocking action of the first dose of the drug. The error produced in the dose-response relation will vary with the size of the safety factor. The size of the safety factor will influence also the latency between the injection of each dose of the drug and the first sign of depressed muscle activity, an effect which may be seen in figure 1 of the communication by Goudsouzian and colleagues (1975). These differences account for the positive results of the present study and the negative conclusions of Goudsouzian and colleagues (1975). The partial loss of safety factor may be regarded as a degree of neuromuscular blockade, insufficient to result in any overt sign of weakness in a recording of twitch tension. It resembles the effect of a small dose of d-tubocurarine which is insufficient to produce any neuromuscular blockade but which renders the
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The two parameters of neuromuscular transmission, quantum size and EPP quantum content, change reciprocally with age (table I). Thus at 30 days a greater quantum size effectively offsets the smaller EPP quantum content, while at 110 days the opposite relationship exists. Deviation from this normal pattern of maturation may produce a reduction in safety factor and the possibility of neuromuscular failure. For example, the characteristic weakness of myasthenia gravis results from a reduction in depolarization produced by each quantum of acetylcholine, and not from a reduction in the EPP quantum content. From microelectrode recording from motor end-plates in human intercostal muscle it has been shown that the mean MEPP amplitude in myasthenic patients was one-fifth of that found in non-myasthenic subjects (Elmqvist et al., 1964). If the same reduction in quantum size were to occur in the 110-day-old rats of this study, the safety factor for the first EPP would be 2.07, while that for the plateau EPP's would be only 0.81. Under such conditions, the muscle fibres of the diaphragm would respond, on average, to only the first and perhaps the second nerve impulses of each train. For succeeding impulses, the EPP amplitude would be below threshold and neuromuscular transmission would fail. In vivo, this sequence of events would curtail the inspiratory action of the diaphragm and so reduce pulmonary ventilation.
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AGE AND NEUROMUSCULAR TRANSMISSION SAFETY FACTOR
in the concentration required to abolish twitching. Small differences in safety factor, such as those between 30- and 110-day-old rats, would not be deteaed in the larger reduoion. This comment may be applied to the study of Goudsouzian and colleagues (1975) and demonstrates the sensitivity of the quantitative methods of measurement of neuromuscular transmission utilized in the present investigation. ACKNOWLEDGEMENT
This study was carried out while S. S. Kelly was an M.R.C. Research Student. REFERENCES
Blackhall, M. I., Buckley, G. A., Roberts, D. V., Roberts, J. B., Thomas, B. H., and Wilson, A. (1969). Druginduced neonatal myasthenia. J. Obstet. Gynaecol. Br. Commonw., 76, 157. Colquhoun, D. (1971). Lectures on Biostatistics. Oxford: Clarendon. Elmqvist, D., Hofmann, W. W., Kugelberg, J., and Quastel, D. M. J. (1964). An electrophysiological investigation of neuromuscular transmission in myasthenia gravis. J. Physiol. {Lond.), 174, 417. Fatt, P., and Katz, B. (1952). Spontaneous subthreshold activity at motor nerve endings. J. Physiol. {Lond.), 117, 109. Goudsouzian, N. G., Donlon, J. V., Savarese, J. J., and Ryan, J. F. (1975). Re-evaluation of dosage and duration of action of d-tubocurarine in the pediatric age group. Anesthesiology, 43, 416. Hubbard, J. I., Llinas, R., and Quastel, D. M. J. (1969). Electrophysiological Analysis of Synoptic Transmission. London: Edward Arnold (Publishers) Ltd. Katz, B., and Thesleff, S. (1957). On the factors which determine the amplitude of the "miniature end-plate potential". J. Physiol. {Lond.), 137, 267. Kelly, S. S. (1976). The effects of age and nutrition on neuromuscular transmission. Ph.D. Thesis, University of Liverpool. Liley, A. W. (1956). An investigation of spontaneous activity at the neuromuscular junction of the rat. J. Physiol. {Lond.), 132, 650. Marshall, M. W., and Ward, M. R. (1974). Anode break excitation in denervated rat skeletal muscle fibres. J. Physiol. {Lond.), 236, 413. Martin, A. R. (1955). A further study of the statistical composition of the end-plate potential. J. Physiol. {Lond.), 130, 114. Redfern, P. A. (1970). Neuromuscular transmission in newborn rats. J. Physiol. {Lond.), 209, 701. L'EFFET DE L'AGE SECURITE DANS NEUROMUSCULAIRE ISOLE
SUR LE FACTEUR DE LA TRANSMISSION SUR LE DIAPHRAGME DU RAT
RESUME
Une analyse de la transmission neuromusculaire a ete faite dans les preparations de nerf phrenique/diaphragme de
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subject more sensitive to a further dose of the drug. In terms of the dose-response curve, a loss of safety factor would be reflected by a shift of the curve to the left and the magnitude of displacement would be equal to the dose of d-tubocurarine which would produce an equivalent reduction in safety factor. It has been shown that d-tubocurarine, in a concentration of 5 x 10~7 w/v, reduced MEPP amplitude and hence, safety faaor, by 50% (Fatt and Katz, 1952). Such a concentration in the plasma could result from a dose of 0.0225 mg/kg given rapidly i.v. into a plasma volume of 45 ml/kg body weight. The concentration of d-tubocurarine at the end-plates would be marginally less, by dilution in the small amount of interstitial fluid between capillary and end-plate. On this basis, for a 50% loss of safety factor there would be a shift in the dose-response curve equivalent to 0.0225 mg/kg of d-tubocurarine. This is of the same order of magnitude as the standard errors of the means of effective doses of d-tubocurarine found by Goudsouzian and colleagues (1975)'and it is unlikely therefore that their method would detect a difference in safety factor similar to that found between 30-day and 110-day-old rats. A displacement of the dose-response curve to the left may provide an explanation for the greater variance in effective doses of d-tubocurarine noted by Goudsouzian and colleagues (1975). The curve is sigmoid in shape and the initial dose of d-tubocurarine 0.1 mg/kg occurs at the lower inflexion of the curve where the response is insensitive to small differences in dose. A displacement to the left would cause the initial dose to lie on a steeper portion of the curve and larger changes in response would occur. This could increase the variance of the lines drawn through the corresponding dose-response values for each subject and lead to a greater range of effective dose values, particularly at the ED 80 and ED 95 levels. In view of the difference in safety factor between 30-day and 110-day-old rats it might be expected that the diaphragms from the young rats would require less d-tubocurarine in the bathing fluid to produce a given degree of neuromuscular block. However, the concentration of d-tubocurarine was varied within the range 10-«+10%w/v (1.15-1.40x 10~6mol/litre) in order to obtain EPPs of optimal size in relation to background noise and facilitate accurate measurements of their amplitudes. There was no obvious difference in the concentration of d-tubocurarine required to produce these conditions in the two groups of rats. This would be anticipated, in view of the large reduction in safety faaor produced by d-tubocurarine
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DER ALTERSEINFLUSS AUF DEN SICHERHEITSFAKTOR IN NEUROMUSKULAREN U B E R T R A G U N G E N IM ISOLIERTEN
RATTENZWERCHFELL ZUSAMMENFASSUNG
Die neuromuskulare iibertragung wurde in Phrenikus/ Zwerchfell-Praparaten aus 30-Tage oder 110-Tage alten Rattenmannchen untersucht. Die Kleinspannungen an den Nervenendplatten wurden als 0,969 ± SFM (Standardfehler des Mittels) 0,058 mV ftir 30 Tage und 0,510 +SFM 0,031 mV fur 110 Tage ermittelt, d.h. ihre Grosse sank mit steigendem Alter ab. Wahrend der gleichen Zeitspanne stieg der Quantengehalt der ersten Nervenendplatten spannung einer 10-Hz Serie von 40 von 144,5 SFM + 11,1 -10,4 auf 346,0 SFM +41,4 -37,0 an. Nach den Beobachtungen anderte sich der Durchschnittsquantengehalt der letzten 30 Nervenendplattenspannungen jeder
Serie dementsprechend: von 50,6 SFM +3,5 —3,2 auf 138,9 SFM +15,0 -13,6. Es wurde festgestellt, dass der aus diesen gemessenen Parametern errechnete Sicherheitsfaktor fur neuromuskulare iibertragung fur 30 Tage nur 70-80% dessen fiir 110 Tage betrug. Es wurde gescha'tzt, dass der in jungen Ratten festgestellte untere Sicherheitsfaktor ungefahr der neuromuskularen Blockierungswirkung einer Mindestdosis von 0,0225 mg/kg des d-Tubocurarins glich. Eine Extrapolation dieser Ergebnisse auf den Menschen wurde fruhere Berichte uber erhohte d-Tubocurarin-Empfindlichkeit in Neugeborenen bekraftigen. EL EFECTO DE LA EDAD SOBRE EL FACTOR DE SEGURIDAD EN LA TRANSMISION NEUROMUSCULAR EN EL DIAFRAGMA AISLADO DE LA RATA SUMARIO
Se ha efectuado un analisis de transmision neuromuscular en preparaciones de nervio frenico diafragma procedentes de ratos de 30 o 110 dias. La amplitud de los potenciales de placas terminales miniatura se hallo que disminula con la edad, siendo 0,969 + SEM (E.T.M.) 0,058 mV a los 30 dias de edad, y 0,510 ±0,031 E.T.M. mV a los 110 dias. Durante el mismo periodo, el contenido quantum del primer potential de placa terminal de una serie de 40 a 10 Hz aument6 desde 144,5 E.T.M. + 11,1,-10,4 hasta 346 E.T.M. + 41,4, — 37,0. Tambito se observ6 un cambio correspondiente en el contenido promedio de quantum de los ultimos 30 potenciales de placa terminal de cada serie; desde 50,6 E.T.M. +3,5, - 3 , 2 hasta 138,9 E.T.M. +15,0, -13,6. El factor de seguridad para la transmisidn neuromuscular, calculado a partir de estos parametros medidos, se hallo que era a los 30 dias solamente el 70-80% del a los 110 dias. Se calculo que el mas bajo factor de seguridad hallado en los ratos jovenes era aproximadamente a la action bloqueadora neuromuscular de una dosis de, por lo menos, 0,0225 mg/kg de d-tubocurarina. La extrapolaci6n de estos resultados al hombre respaldaria anteriores comunicaciones sobre una mayor sensibilidad a la d-tubocurarina en los neonatos.
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rats males Sges de 30 jours ou de 110 jours. On a trouve que l'amplitude des potentiels des plaques d'extr&nite miniaturisees allait en decroissant avec l'^ge, 6tant de 0,969 + SEM (erreur standard des moyennes) 0,058 mV a 30 jours et de 0,510 ± SEM 0,031 mV a 110 jours. Au cours de la meme periode la teneur du quantum du potentiel de la premiere plaque d'extremite d'une serie de 40 a 10 Hz a augmente de 144,5 SEM + 1 1 , 1 , - 10,4 a 346 SEM +41,4, — 37,0. Une variation correspondante a egalement 6te observee dans les teneurs du quantum moyen des derniers 30 potentiels de plaque d'extremite de chaque serie; de 50,6 SEM +3,5, - 3 , 2 a 138,9 SEM +15,0, -13,6. On a trouve que le facteur de securite pour la transmission neuromusculaire, calcule a partir de ces parametres mesures, n'etait a 30 jours que de 70-80% de celui a 110 jours. On a estime que le facteur de securite plus bas, que Ton a trouve sur les jeunes rats, etait a peu pres equivalent a l'action de blocage neuromusculaire d'une dose d'au moins 0,0225 mg/kg de d-tubocurarine. L'extrapolation de ces rdsultats pour rhomme permet d'appuyer les rapports precedents sur la sensibilite accrue des nouveaux-nes a la d-tubocurarine.
