51

J. Physiol. (1978), 274, pp. 51-62 With 6 text-ftgure'8 Printed in Great Britain

THE EFFECT OF AGE ON NEUROMUSCULAR TRANSMISSION

BY S. S. KELLY

From the Department of Physiology, University of Liverpool, Liverpool

(Received 18 April 1977) SUMMARY

1. Resting membrane potentials (RMPs), spontaneous miniature end-plate potentials (m.e.p.p.s), and evoked end-plate potentials (e.p.p.s) were recorded in phrenic nerve-hemidiaphragm preparations from rats at ages from 11 to 375 days. 2. The mean RMP increased from - 6441 + 1P2 mV (mean + s.E.) at age 11 days to - 71-3 + 1 0 mV at age 30 days, after which there was no significant change with age. 3. The mean amplitude of m.e.p.p.s decreased from 1P088 + 0'070 mV at 11 days of age to 0 405 + 0 030 mV at 175 days of age, after which there was no significant change. 4. There was a rapid, large increase in the frequency of m.e.p.p.s from 0-02/sec to 0.97/sec (geometric means) between 11 and 23 days of age, followed by a slower increase to 3-19/sec at 175 days of age. Subsequently there was a decrease to 2*58/sec at 375 days of age. 5. The mean quantum content of plateau e.p.p.s elicited at a frequency of 10 Hz increased from 20-5 quanta/e.p.p. to 169-9 quanta/e.p.p. (geometric means) between 11 and 175 days of age and then decreased to 120X4 quanta/e.p.p. at 375 days of age. 6. The mean quantum content of the first e.p.p.s of trains of e.p.p.s increased from 44*2 quanta/e.p.p. to 468 8 quanta/e.p.p. (geometric means) between 11 and 175 days of age and then decreased to 358-1 quanta/e.p.p. at 375 days of age. 7. The calculated safety factor of neuromuscular transmission increased with age up to 110-175 days and subsequently decreased. 8. The change in all the above parameters occurred most rapidly in the first 6 -weeks of life. The rapidity of these changes indicates that great care must be taken to ensure that control and experimental animals are adequately matched according to age, especially when rats weighing less than about 300 g are used. INTRODUCTION

There has been no systematic investigation of the changes which occur in neuromuscular transmission in rats during the first year of life, although the development of neuromuscular transmission in foetal and neonatal rats was investigated by Diamond &; Miledi (1962), who observed changes in m.e.p.p. amplitude and frequency. They found that the frequency of m.e.p.p.s recorded from neonatal rats was approximately 100 times less than that observed in the adult and that the m.e.p.p. amplitudes in neonates were greater than those of mature animals. The amplitudes and frequencies of m.e.p.p.s recorded in the levator ani muscles of rats aged 2 months and 30 months were studied by Gutmann, Hanlikova & Vyskocil (1971) who found that the

S. S. KELLY amplitude in the older animals was greater than that in the younger animals, but that this was not due to a change in the sensitivity of the muscle fibres to acetylcholine. Gutmann et al. also found that the frequency of m.e.p.p.s was lower in the older animals. The frequency of m.e.p.p.s in the soleus and diaphragm muscles of the rat has been compared in animals aged 3 and 30-33 months by Vyskocil & Gutmann (1972), who found that the m.e.p.p. frequency was lower in the older animals than in the younger animals and that the difference was greater in the soleus than in the diaphragm. This study was carried out in order to observe more closely the changes in m.e.p.p.s and e.p.p.s which occur in the rat phrenic nerve-diaphragm preparation during this period of growth. Many rats of about 200 g body weight are used in experiments, and albino male rats show a rapid increase in body weight (approximately 7 g/day) at that stage of growth. If parameters of neuromuscular transmission change in a similarly rapid manner, a detailed knowledge of those changes would aid in the design of experiments and the interpretation of experimental results. 52

METHODS

Preparation and recording. The rats used in these experiments were albino males, strain CFHB, of known age supplied by Anglia Laboratory Animals. The animals were used in specific age groups, the stated age value of a group representing the mean age of that group. All the rats in the younger age groups were within 1 day of the stated mean age for that group, and rats in groups older than 56 days were within 2 days of the group mean. There were between three and six animals in each group. Two segments of diaphragm, together with their phrenic nerve supplies, were removed from animals under ether anaesthesia. Immediately after removal the segments of diaphragm were placed in oxygenated saline at room temperature and pinned on to a polyvinyl chloride and Perspex base which allowed the end-plate region of the preparation to be transilluminated. Preparations were bathed in Liley saline through which a 95 % 02/5 % CO2 gas mixture was bubbled for at least 1 hr before use, and the composition of this saline was: NaCl, 136-8 mM; KCl, 5-0 mM; CaCl2, 2-0 mM; MgCl2, 1-0 mM; NaHCO2, 24-0 mM; NaH2PO4, 1 0 mM; D-glucose, 10 mm; choline chloride, 10-6 g/ml. In order to record e.p.p.s, neuromuscular transmission was blocked by the addition of (+ )-tubocurarine chloride (10-6 g/ml.) to the saline. The preparation was placed in the upper chamber of a double chamber Perspex bath through which oxygenated Liley saline flowed at a rate of about 0 5 ml./sec, which was sufficient to change the bathing medium in the bath about 1-5 times/min. Warm water was circulated through the lower chamber in order to maintain the preparation bathing medium at a temperature of 32-0 + 0 5 0C. Glass capillary micro-electrodes filled with 4 M-KCl solution were used to record m.e.p.p.s and e.p.p.s intracellularly. Resting membrane potentials and end-plate potentials were observed on a Tektronix type 502A dual beam oscilloscope and recorded using a Siemens oscillomink ink recorder. The shapes and rise times of end-plate potentials were measured from traces on a Telequipment type DM53A dual beam storage oscilloscope. End-plate potentials were regarded as focal and recorded only if their rise times were less than 1 msec. The phrenic nerve was stimulated by means of a Grass S 88 Stimulator and a Grass SIU 4678 stimulus isolation unit using either silver hook electrodes or a suction electrode. At least 15, and occasionally up to 200 focal m.e.p.p.s were recorded at each end-plate, and the micro-electrode was moved from muscle fibre to muscle fibre across the preparation to ensure that recordings were not made more than once from the same end-plate. The preparation was then perfused with saline containing (+)-tubocurarine and allowed to equilibrate with this solution for 30 min, after which time stimulation of the phrenic nerve at a frequency of 10 Hz was found to produce e.p.p.s between 0-5 and 2-0 mV in amplitude. Two to four trains of 40 e.p.p.s at a frequency of 10 Hz were recorded at each end-plate, with an interval of at least 1 min

AGE AND NEUROMUSCULAR TRANSMISSION

53

between trains. Preliminary experiments, in which several trains of e.p.p.s were elicited at the same end-plate, showed that a 1 min interval was sufficient to allow for full recovery of neuromuscular transmission. Calculations and statistics. The amplitudes of both spontaneous and evoked end-plate potentials were corrected for non-linear summation of depolarization by the subsynaptic membrane (Martin, 1955). End-plate potentials were corrected to a standard RMP to facilitate the comparison of m.e.p.p. amplitudes from different muscle fibres and from different groups of rats. The value of the standard RMP affects the values of the mean amplitudes of m.e.p.p.s and e.p.p.s, but it has no effect on the values of calculated quantum contents of e.p.p.s. The chosen value for the standard RMP was -71 mV, the mean value for all groups of rats over 23 days of age. The corrections were made by applying the following equation

V_V

V'.(71-15)

(RMP -15 -V')'

(1) 1

where V is the corrected amplitude (mV), V' is the uncorrected amplitude (mV), and - 15 mV was taken as the acetylcholine reversal potential (Takeuchi & Takeuchi, 1960). The true value of the reversal potential was not measured in this study, but calculations showed that varying the reversal potential from -20 to 0 mV made less than 5 % difference to the parameters investigated in this study. Although Martin's correction is likely to be inaccurate when applied to large end-plates potentials (Martin, 1976; Stevens, 1976), the largest of the e.p.p.s recorded in this investigation, i.e. the first e.p.p.s of trains, was less than 7 mV; thus the correction was small and any inaccuracy involved would not affect the validity of these results. The mean corrected amplitude of m.e.p.p.s at each end-plate was calculated and from these mean values the mean, standard deviation (S.D.), and standard error (s.E.) of the mean for each age group was calculated. The distributions of the amplitudes of m.e.p.p.s at any one end-plate, the distributions of mean m.e.p.p. amplitudes between end-plates in any one group, and the distributions of RMPs in any one group were found to conform to a Gaussian distribution when the x-squared test was applied. All the other parameters included in this study were found to differ significantly from a Gaussian distribution but not from a log-normal distribution. The mean and standard deviations of each of the parameters whose distributions conformed to a Gaussian distribution were calculated in the usual manner, but the values of the other parameters were converted to logarithms before the means and standard deviations were calculated to provide geometric means and standard deviations. When calculating the quantum content of plateau e.p.p.s, i.e. the last 30 e.p.p.s of trains of 40 e.p.p.s, a Poisson distribution of quantum contents was assumed (Boyd & Martin, 1956; Elmqvist & Quastel, 1965) so that V2

var V

(2)

where V is the mean corrected e.p.p. amplitudes, var V is the variance of the corrected e.p.p. amplitudes, and m is the mean quantum content of the e.p.p.s. However, the variance of the quantum units and the recording noise contributed to the over-all measured variance of the e.p.p.s, and for this reason a correction was applied using measured values for the variance of quantum units (assuming the coefficient of variation of the quantum units was the same as the coefficient of variation of the m.e.p.p.s) and for the variance due to noise (cf. Miyamoto, 1975). This correction is shown in eqn. (3). V72 =1 + CVM,) X (3) var V-var (n) where CVm is the coefficient of variation of m.e.p.p.s and var (n) is the variance due to noise. Measured values for C Vm and var (n) obtained from different groups of rats were found to be not significantly different. The value of the quantum content of the first e.p.p. of a train was calculated from the ratio of the corrected amplitude of the first e.p.p. to the mean corrected amplitude of the plateau e.p.p.s, and this ratio was multiplied by the calculated mean quantum content of the plateau e.p.p.s. The mean value of these parameters were calculated for each end-plate, and the geometric mean and the S.D. of the logarithms of the end-plate means were calculated for each age

S. S. KELLY

54

group. Student's t test was then applied to the mean and S.D.s of the logarithms to determine the significance of any difference between age groups. Calculation of safety factor. The safety factor of neuromuscular transmission was defined, for the purposes of this investigation, as the depolarization which could be produced by transmitter released by the motor nerve terminal divided by the minimum depolarization required to initiate an action potential in the muscle fibre. The threshold level of depolarization required to initiate an action potential in fibres from rats aged 56 days was found to be -56 mV, a value similar to that of -53-45 ± 039 mV obtained by Marshall & Ward (1974). The depolarization required to reach the threshold level would be (RMP -56) mV, and the mean depolarization which would be produced by the e.p.p.s in the absence of (+ )-tubocurarine was taken to be the product of the mean m.e.p.p. amplitude and the mean quantum content for each group of rats. It is necessary, however, to correct for non-linear summation of depolarization by the subsynaptic membrane, and for this purpose eqn. (1) was applied to the depolarization required to reach the threshold level. Thus, if the RMP was -71 mV, to depolarize the membrane by 15 to -56 mV would require the summation of 21 unit depolarizations of 1 mV. The safety factor was taken as the product of the mean m.e.p.p. amplitude and mean quantum content divided by the corrected amount of depolarization required to reach the threshold level. The coefficient of variation of the safety factor, C V8F, was calculated from the coefficient of variation of the m.c.p.p.s, CV., and the coefficient of variation of the e.p.p. quantum contents, CVe, as shown in eqn. (4),

(cf. Colquhoun, 1971).

C T72F

=

C V2+C V+(CVM. C V).

(4)

RESULTS

Body weight. It may be seen from Fig. 1 that there was a rapid increase in body weight with age in the first few weeks of life and that up to about 56 days of age (ca. 300 g) the weight of the animals was a reliable guide to age. Animals older than 100 days showed much greater variation in body weight and it was observed during dissection that this appeared to be associated with the amount of fatty tissue in the thorax and abdomen. Thus, over 300 g weight, the growth rate is much slower and the body weight of an animal becomes progressively less reliable as a guide to age and degree of development (Table 1). Resting membrane potential. The mean RMPs of the 11 and 23 days old groups of rats were significantly lower (5 % < P < 10 %) than the mean RMPs of any of the other groups of rats (Table 2). After 30 days of age the mean RMP did not change significantly at least up to 375 days of age, except for that of the 56 days old animals which was significantly higher than that of any other age group (5 % < P < 10 %). M.e.p.p. amplitude. The mean amplitude of m.e.p.p.s was found to decrease by more than 50 % from 1 061 to 0'405 mV between the ages of 23 days and 175 days (Fig. 2), after which there was no further change with age at least up to 375 days of age. There was no significant difference between the mean m.e.p.p. amplitudes of the 11 and 23 days old groups of animals (P > 10%) when Student's t test was applied to the mean and S.D.S of the m.e.p.p. amplitudes either with or without correction to the standard RMP of -71 mV. M.e.p.p. frequency. A large increase in m.e.p.p. frequency from 0-02/sec to 0.97/sec (geometric means) occurred between 11 and 23 days of age, after which there was a lower rate of increase to 3.19/sec at 175 days of age. At 262 and 375 days of age the m.e.p.p. frequency had decreased to 2.67/sec and 2.58/sec respectively (Fig. 3). Quantum content of plateau e.p.p.s. The mean quantum content of plateau e.p.p.s elicited at a frequency of 10 Hz increased from 20 5 quanta/e.p.p. at 11 days of

AGE AND NEUROMUSCULAR TRANSMISSION

55

600

I

I

I

0

-c

0,

4-

300 .

._5

0 0

0

0

_

I

I

10

20

I

.I

4 400

I

40 100 Age (days)

200

Fig. 1. The relationship between rat body weight and age. Points represent the means of three to six rats. Standard errors of the means are within the filled circles up to 175 days of age, but may be seen in Table 1. The logarithmic age scale is for convenience of plotting only. TABLE 1. The relationship between body weight and age. n is the number of animals in each group

Age (days) 11 23 30 44 56 110 175 262 375

Mean weight (g) 28 60 103 205 298 435 510 511 567

S.E.

1 2 2 3 5 3 16 11 17

TABLE 2. Effect of age on resting membrane potential. n Age (days) 11 23 30 44 56 110 175 212 375 *

Mean RMP (mV) *64.1 *68.5 71*3 71*7 *73.9 70*4 70*1 70-2 71-1

n 4 4 5 5 6

(g)

=

3 3 3 6

number of muscle fibres

S.E. (mV)

1-2 0-8

22 29 33 28 36 33 36 44

1.0 0-9 1.0 0-9 08 0-7 0o5

Values which differ significantly from any of the other values (P

91 =

5% < t < P

=

10%

).

56

S. S. KELLY

1.2 r-

I

E -o0

a. E 06I co

I

6. 6.

Q)

o

L

I

I

10

20

I

I

I__

40 100 200 400 Age (days) Fig. 2. The relationship between m.e.p.p. amplitude and age. Each point represents the mean (± 1 s.E.) of the results from end-plates of each age group of three to six rats. Logarithmic age scale for convenience of plotting only. 4 .-I

C)

U,

C.)

I

C1)

dy

I

204I0

2

C1) 6-

4i 6.

oL

1 I

10

20

I

40 100 Age (days)

-

200

400

Fig. 3. The relationship between m.e.p.p. frequency and age. Each point represents the geometric mean (± 1 s.E.) of the results from end-plates of each age group of three to six rats. Logarithmic age scale for convenience of plotting only.

age to 169-9 quanta/e.p.p. at 175 days of age (geometric means). After 175 days of age there was a slow decrease to 120-4 quanta/e.p.p. at 375 days of age (Fig. 4). Because the product of the mean plateau e.p.p. quantum content and the stimulus frequency is equivalent to the rate of mobilization of quanta of transmitter, this parameter also changes in the same manner as the mean plateau e.p.p. quantum content. Quantum content of the first e.p.p. of a train of e.p.p.s. The geometric mean quantum content of the first e.p.p.s of trains increased with age from 44-2 quanta/e.p.p. at 11 days of age to 468-8 quanta/e.p.p. at 175 days of age, after which there was a gradual decrease in quantum content to 358-1 quanta/e.p.p. at 375 days of age (Fig. 5).

AGE AND NEUROMUSCULAR TRANSMISSION 57 Safety factor of neuromuscular transmission. A small but significant increase with age was found when both plateau e.p.p. and first e.p.p. safety factors were calculated (Fig. 6). The safety factor of mean plateau e.p.p.s elicited at a frequency of 10 Hz increased from 2*02 at 11 days of age to 3*61 at 110 days of age, and the safety factor of the first e.p.p. increased from 4.35 to 9-87 between 11 and 175 days of age.

160 r

If II 0~ lco Co.

80 F

I I I

K 0L

I

I

I

10

20

I

I

40 100 Age (days)

-

200

400

Fig. 4. The relationship between the mean quantum content of plateau e.p.p.s elicited at a frequency of 1OHz and age. Each point represents the geometric mean ( ± 1 s.E.) of the results from end-plates of each age group of three to six rats. Logarithmic age scale for convenience of plotting only. 500 r

I

to -

co

0_

o. In 250

I

0. U,

I

II

I

I

I

I 0L

I

I

10

20

I

I

40 100 Age (days)

I

200

400

Fig. 5. The relationship between the quantum contents of the first e.p.p. of trains of e.p.p.s and age. Each point represents the geometric mean (± 1 s.E.) of the results from end-plates of each age group of three to six rats. Logarithmic age scale for convenience of plotting only.

S. S. KELLY

58 10

on

05

I

I

10

20

40 100 Age (days)

I

200

I

400

Fig. 6. The relationship between calculated safety factor and age. Points represent the geometric mean safety factor (± 1 s.E.). Open circles: safety factors of mean plateau e.p.p.s elicited at a frequency of 1O Hz; filled circles: safety factors of the first e.p.p. of trains of e.p.p.s. A safety factor of 1-0 is just sufficient to allow neuromuscular transmission. The increase in safety factor between 11 and 175 days of age was significant (P < 5 %, t test) as was the decrease in safety factor between 175 and 375 days of age. Logarithmic age scale for convenience of plotting only. DISCUSSION

One possible explanation for the low RMPs observed in the 11 and 23 days old rats might be that more damage was done to muscle fibres with smaller diameters by micro-electrode penetration. If this were so, one would expect the coefficient of variation of the RMPs of each of these two age groups to be considerably greater than those of older rats because the degree of damage caused by the micro-electrode would add an extra degree of variability to the measurements, but the coefficient of variation was found to remain unchanged. If damage by the micro-electrodes was responsible for the low RMPs in young rats, it might be expected that, by chance, some fibres would be relatively undamaged and have RMPs as high as the highest values found in older animals. The five highest values recorded in the 11 days old group wereS 77, 73, 72, 71 and 66 mV (mean = 71-8 mV; S.D. = 4-0 mV), and the five highest values recorded in the 30 days old group were: 90, 84, 83, 81 and 80 mV (mean = 83-6 mV; S.D. = 3-9 mV). Thus it seems unlikely that the low RMPs of the 11 and 23 days old rats were caused solely by micro-electrode damage. The high RMP of the 56 days old group could be due to variability of samples since statistically the difference is barely significant, but could not be due to differences in experimental conditions, which were the same for all groups of rats. M.e.p.p. amplitude. The most likely cause of the decrease in m.e.p.p. amplitude

AGE AND NEUROMUSCULAR TRANSMISSION 59 with age is an increase in the diameter of muscle fibres rather than an increase in cholinesterase activity, because the time courses of the m.e.p.p.s from young rats were not measurably different from those of older animals (the rise times being about 0 5 msec and half decay times about 1P0 msec), and it is known that anticholinesterases increase the time courses as well as amplitudes of end-plate potentials (Fatt & Katz, 1951). Further evidence to support this suggestion is that Katz & Thesleff (1957), using frog sartorius preparations, found that m.e.p.p. amplitude was inversely related to muscle fibre diameter and in mice (Rowe & Goldspink, 1969) and humans (Moore, Rebeiz, Holden & Adams, 1971), muscle fibre diameter has been found to increase with age until a plateau level is reached. Other factors which may have caused or contributed to the decrease in m.e.p.p. amplitude with age are a decreased amount of acetylcholine per quantum or a change in the properties of the ion channels opened by acetylcholine. The most rapid period of change of m.e.p.p. amplitude with age occurred during the most rapid growth period (23-56 days of age), and after 175 days of age there was only a small increase in body weight and no significant change in m.e.p.p. amplitude. However, between the ages of 11 and 23 days of age there was no significant change in m.e.p.p. amplitude, while there was an approximate doubling of body weight. This could have been brought about by increase in number, rather than diameter, of muscle fibres, or by a change in the input resistance of the muscle fibres, or by a coincident increase of quantum size with the increased muscle fibre diameter, perhaps due to an increased amount of transmitter released per quantum, or due to an increase in the sensitivity of the post-synaptic membrane. The increase in m.e.p.p. amplitude with age between 3 and 30 months old rats observed by Gutmann et al. (1971) indicates that the m.e.p.p. amplitude increases again between 12 and 30 months of age. M.e.p.p. frequency. There was a significant increase in m.e.p.p. frequency with age, despite the observation that there was often a tenfold difference of mean frequencies between end-plates in the same group of animals. The greatest change in m.e.p.p. frequency occurred between 11 and 23 days of age, during which period there was an approximately 60-fold increase. A similar large increase in m.e.p.p. frequency between neonatal and mature rats was observed by Diamond & Miledi (1962). They considered that the low m.e.p.p. frequency in young rats was not due to a lack of quanta available for release because stimulation of the motor nerve considerably increased transmitter release; they suggested that the lower frequency in young animals was related to the smaller area of synaptic contact at the neuromuscular junctions of neonates (Kelly & Zacks, 1969a, b). Plateau e.p.p. quantum content. The quantum content of e.p.p.s at the plateau will be independent of the variable 'store' and 'probability of release', because at the equilibrium state the rate of release of quanta must equal the rate at which quanta are made available for release, i.e. mobilization. It has been found (Tucek, 1972) that the activity of choline acetyltransference in the rat diaphragm increases during postnatal development. In these experiments, however, trains of only 40 e.p.p.s were elicited with at least 1 min between trains, and, under such conditions, it might be expected that the rate of mobilization of quanta would not be limited by the synthesis or packaging of transmitter. If it is postulated that the mobilization rate is

60

S. S. KELLY

related simply to the growth of the nerve terminal, it becomes difficult to explain the decrease in mobilization rate between 175 and 375 days of age unless there is also some shrinkage of the nerve terminal. First e.p.p. quantum content. The first e.p.p. of a train of e.p.p.s can be considered as a single sample of the population of quanta in the nerve terminal, i.e. mo = p.N, (5) where mo is the quantum content of the first e.p.p.s, p is the fractional release (probability), and N is the number ofquanta available for release. It follows, therefore, that the observed increase in mo with age could be due to an increase in either p, or N, or both. Kuno, Turkanis & Weakley (1971) found that the quantum content of e.p.p.s was proportional to the end-plate area at the frog sartorius neuromuscular junction, so that an increase in N might be expected to be related to an increase in the size of the nerve terminal as the animal grows. However, the subsequent small but significant decrease in mo between 175 and 375 days of age might be difficult to explain on this basis. Safety-factor. The safety factor of neuromuscular transmission increases with age, despite a decrease in quantum size of more than 50 % over the age range studied; the increase in quantum content more than compensates for the decrease in quantum size. Bush & Stead (1962) observed that human neonates are more sensitive to the effects of a given dose of (+ )-tubocurarine per unit body weight than adults, and, provided that the results obtained from rats in this investigation are applicable to humans, this could be explained by an increase in safety factor with age. In order to block neuromuscular transmission, a sufficiently large dose of ( + )-tubocurarine must be given to reduce the quantum size to such an extent that the safety factor falls below a value of one; hence the greater the initial safety factor, the greater the dose of (+ )-tubocurarine which must be given before any block of transmission occurs. In humans in vivo the higher sensitivity of neonate to (+ )-tubocurarine could be due to factors other than any possible alteration in safety factor, such as rate of metabolism or excretion of the drug. Whether or not the observed decrease in safety factor after 175 days of age continues beyond 375 days of age is uncertain because m.e.p.p. amplitude has been observed to increase between the ages of 3 and 30 months (Gutmann et al. 1971), and this may counteract any further decrease in the quantum content of plateau e.p.p.s or the first e.p.p.s of trains. Paton & Waud (1967) used a different method of estimating the margin of safety of neuromuscular transmission when they studied in vivo the tibialis anterior and sartorius muscles of the cat. Their method involved the determination of the fractional occupation of acetylcholine receptors by antagonists when the height of the twitch in response to indirect stimulation at a frequency of 0.1 Hz began to decrease. They calculated that approximately 75 % of the receptors could be occupied by an antagonist before any decrease in twitch height occurred and that an occupancy of about 92 % was required for a 'nearly complete' neuromuscular block. Thus the neuromuscular junctions with the lowest margin of safety which could function with only 25 % of their receptors available to combine with acetylcholine were said to have a value of 4 as their margin of safety and, similarly, those which could function

61 AGE AND NEUROMUSCULAR TRANSMISSION with only 8 % of their receptors available were said to have a value of 12 as their margin of safety. The values for the margin of safety obtained by Paton & Waud, therefore, represent the maximum and minimum values of the population of endplates in each muscle. Although the values obtained in the present investigation of the safety factor of the first e.p.p.s of trains fall within the same range as the values of the margin of safety obtained by Paton & Waud, the two sets of results are not comparable because they were obtained from different muscle preparations from different animals by completely different methods. It is not possible to equate cats of the weights used by Paton & Waud with rats of known age and, even if it were possible, it is unlikely that the margin of safety as defined by them is the same parameter as the safety factor as defined in this study because they measured spare receptor capacity, not spare transmitter capacity as in this study. The safety factor of neuromuscular transmission will be determined by that part of the transmission process which has the smallest margin of safety. Thus, if there are far more receptors than are needed to combine with the maximum amount of transmitter released, as is the case in this investigation where m.e.p.p.s were measured and used to calculate the mean quantum size in unblocked preparations, then the safety factor of transmission will depend upon the amount of transmitter released rather than the number of receptors available. The calculations of safety factor in the present investigation are based on the assumption that the threshold potential remains constant. If this assumption were true, the actual value of the threshold potential would alter the absolute value of the safety factor obtained but would not affect the changes observed to occur with age. Unfortunately, until more evidence is collected to confirm or disprove this assumption, it would be unwise to place too much reliance on the conclusions based upon it. Conclusion. Certain parameters of neuromuscular transmission in rats undergo large changes during the first year of life, and many of these changes may be explained in terms of the growth of the muscle fibres and nerve terminals. Because of these changes, experiments should be designed using adequately age-matched control animals, and care must be taken especially when the animals are in their fast growth phase or when the trauma of a chronic experiment may alter that growth rate. The results obtained in this study indicate that some of the changes which occur up to 175 days of age are reversed as rats age further, but that the changes after this age take place much more slowly. This investigation was carried out whilst the author was in receipt of a Medical Research Council Research Studentship. I would also like to thank Dr D. V. Roberts for advice and encouragement throughout this study. REFERENCES

BOYD, I. A. & MARTIN, A. R. (1956). The end-plate potential in mammalian muscle. J. Physiol. 132, 74-91. BUSH, G. H. & STEAD, A. L. (1962). The use of D-tubocurarine in neonatal anaesthesia. Br. J. Ana&9th. 34, 721-728. COLQUEOUIN, D. (1971). Lectures on Biostatitics, 1st edn., p. 40. Oxford: Clarendon. DIAMOND, J. & MILEDI, R. (1962). A study of foetal and new-born rat muscle fibres. J. Physiol. 162, 393-408.

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The effect of age on neuromuscular transmission.

51 J. Physiol. (1978), 274, pp. 51-62 With 6 text-ftgure'8 Printed in Great Britain THE EFFECT OF AGE ON NEUROMUSCULAR TRANSMISSION BY S. S. KELLY...
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