Acta physiol. scand. 1978. 103. 132-143 F r o m the Departments of Clinical Neurophysiology a n d Neurology, Karolinska Hospital, Stockholm a nd the Department of Ophthalmology, University Hospital, Linkoping, Sweden
The postnatal development of the inferior oblique muscle of the cat I. Isometric twitch and tetanic properties BY GUNNAR
LENNERSTRAND and JERKER HANSON Received 28 November 1977
Abstract LENNERSTRAND, G. and HANSON, J. The postriatal dewlopmerit of the inferior oblique muscle of the cLit. I. Isometric fwilch arid tetnriic properties. Acta physiol. scand. 1978. 103. 132-143. The posrnatal development of the inferior oblique muscle in the cat has been studied with physiological and hictochemical tecknique5. This paper describes the changes with age in isometric twitch and tetanic reymnse characteristics. The twitch amplitude increased and the twitch contraction time (cr) and halfrelaxation time (hrr) decreased almost linearly from birth to adulthood. The relation between the strength of n e n e stimulation and twitch cr and / t r / changed during development with a threshold slow response appearing at 10 weeks. Twitch responses in cats 6 weeks of age or older were of longer duration than in younger c a ts in spite of the longer c f and hrf in young cats. Fusion frequency of the tetanic response reached a constant level in muscles 6 weeks or older. The maximum rate of tension rise remained the same from birth to 6 ueeks of age and later increased markedly up to 20 weeks of age. The contracture induced by succin\Icholinc was the same in muscles of all ages. These data were related to previous findings on the postnatal development of fast and slow muscles and motor units in the hindlimb of the cat. A differentiation of the de\elopment of fast and slow eye muscle fibrcs is suggested. Slow fibres seemed to have completed their niaturation a t about ten wceks of age, while the development of fast fibre properties continued, probably u p ro the adult stage. Kc?, wordv: Postnatal de\elopment, cat extraocular muscle, fast and slow fibres, contractile properties, twitch aiid tetanus
structure, adult e y e muscle is known t o differ markedly from other muscles. The inferior oblique muscle of the cat has been shown to contain si n g l y (focally) innervated muscle fibres of t h e same t y p e s t h a t exist in mammalian skeletal muscles (Alvarado and van Horn 1975), but in addition m u lti p l y innervated fibres, not seen in ordinary skeletal muscle, h a v e been identified (Hess and Pilar 1963, Peachey, Takeichi and Nag 1975, Alvarado and van Horn 1975, Mayr 1975). The multiply innervated fibres are of 2 types, one of t h e m resembling slow fibres in amphibian muscle and t h e o t h e r slow f ibr e s in avian muscle (Peachey 1971, Peachey et a!. 1975, Alvarado and van Horn 1975, M a y r 1975). W i t h r e g a r d t o muscle fibre
132
TWITCH AND TETANUS IN KITTEN EYE MUSCLE
I33
Three types of motor units with different functional properties have been identified in the cat inferior oblique muscle (Lennerstrand 1974, 1975). The units with the fastest contractions are probably composed of singly innervated fibres and the slowest units of multiply innervated fibres of the slow amphibian type. The units of intermediate speed of contraction have been suggested to consist of multiply innervated fibres resembling the slow fibres in avian muscle (Bach-y-Rita and Ito 1966, Lennerstrand 1974, 1975, Lennerstrand and Bach-yRita 1974, Bach-y-Rita 1975). It is not known how the properties of eye muscle fibres in the cat change from birth to the adult stage. Matyushkin (1967) reported that the contraction of eye muscles in young rabbits were slower than those of adult muscle, and similar observations have been made on rat eye muscle by Close (1975). In recent years the postnatal development of skeletal muscle in the cat has been extensively studied, particularly the functional differentiation of the fast and slow twitch fibre systems in hindlimb muscles (Buller, Eccles and Eccles 1960, Buller and Lewis 1965, Close and Hoh 1967, Mann and Salatsky 1970, Bagust, Lewis and Westerman 1973, Westerman et al. 1973, Hammarberg and Kellerth 1975 a, b). At birth all hindlimb muscles contract slowly. They seem to be composed of the same types of fibres (Nystrom 1968 b, Maier and Eldred 1974, Hammarberg 1974). During the first postnatal weeks a differentiation takes place, and some muscles like the gastrocnemius increase their speed of contraction with age, while others like the soleus remain slow throughout development (Buller et al. 1960, Buller and Lewis 1965, Nystrom 1968 a, Westerman et al. 1973). The differentiation of fast and slow contraction seems to occur in parallel with changes in fibre composition (Nystrom 1968 b, Maier and Eldred 1974, Hammarberg 1974). The purpose of the present investigation has been to study the postnatal development of cat extraocular muscle with physiological and histochemical techniques. A comparison with the development of fast and slow fibre systems of other cat muscles will be attempted although this will be somewhat difficult due to the lack of information on the postnatal development of muscle fibres with multiple innervation, even in amphibian and avian muscle. Isometric contractions have been recorded in the inferior oblique muscle of kittens of different age and in the adult cat. It has not been possible to record from individual fibres or motor units, and conclusions have to be drawn from data obtained from the whole muscle. In this paper the postnatal changes in twitch and tetanic conctraction will be evaluated. In a subsequent paper the changes with age in fatigue properties and other post-tetanic effects will be examined (Lennerstrand and Hanson 1978). The developmental changes in the histochemical fibre spectrum of the inferior oblique muscle will be described separately (Hanson, Nichols and Lennerstrand 1978). Preliminary reports on the results have been published elsewhere (Hanson, Lennerstrand and Nichols 1977, Lennerstrand, Hanson and Nichols 1977).
Methods 25 kittens and 3 adult cats were used in the study. They were arranged in 8 groups according to postnatal age. The number of animals in each group and their body weight are shown in Table I. In most animals muscles of both eyes were used for experiments. The animals were anesthetized with pentobarbital (NembutaP) 40 mg/kg b.w. injected iritraperjtoneally and the trachea and one femoral vein were cannulated. Additional doses of anesthetic were given i.v. when needed. The head was rigidly clamped with earpins and a bite bar. The inferior oblique, the lateral and
I34
GUNNAR LENNERSTRAND AND JERKER HANSON
T A M E1. Presentation of the groups of experimental animals in the study. Group 0 weeh 1 weeh
Age (days) 2-3 6-9
2 week\
12-17
4 weeks
29-32
6 \reel\,
4245 66-74 123. 1 5 1 over 365
10 v,eeks
20 Heehs Adult
Weight (g)
Animals
Muscles
138-143 195-26 1 22 1-326 308487 348-550 600-900 650. I 200 2 800-3 500
medical recti and the retractor bulbi muscles were freed from the globe, which was enucleated. The nictitating membrane was raised to form a pool in the orbit, which was filled with warm mineral oil (35-37°C). The bod! temperature was monitored continuously and regulated to 37-38'C with a beating pad. After the experiments parts of the muscles were removed for histochemistry. /.\or?ietrir rcnsroti recording. The short tendon of the inferior oblique muscle was tied with braided sutures to a hook glued to one end of a sensitive strain gauge (Endevco no. 8101). The other end of the strain gauge was cldniped to a metal base attached to a micromanipulator. The undamped reasonance frequency in air of the tension recording assembly was 2 kHz. The linear range of the system spanned froni 10 mg to 40 g. The signals from the strain gauge \\ere amplified and displayed o n a Medelec UV recording unit. The passhand of the amplifiers was set at 0 4 0 kHz. In order to enable comparison between contractions in different muscles, a11 tension recordings were made at the muscle length of maximal twitch response. Care was taken to align the strain gauge perpendicular to the natural pulling direction of the muscle. Stitiiihriorr. The muscle nerve was carefully dissected free from orbital tissue and cut as far centrally 3 s possible. I t w a s positioned over paired platinum electrodes and stimulated with square-wave pulses of 0.1 n i \ duration. generated by a Grass S8 stimulator and fed through an isolation unit. R ~ o r d r r i parid r ~ i ~ ~ u . ~ i ~ r ~ With ~iii~~ some r i t . \ ,slight modifications the techniques have been similar to those used by Haiison and Lennerstrand (1977). Twirclr rrspumcs were recorded first to increasing intensities of single pulse stimulation from threshold to supramaximal values. Threshold was about 0.4 V for the newborn animals and decreased to about 0.2 V for adult cats. Stimulus intensities at least five times those for rnawnial raitch response were used for the res: of the recordings of twitch and tetanic responses. The maximal amplitude of the twitch, the contraction time ( r t ) and the half-relaxation time (hrt) of the twitch contraction were measured. I n addition the time for complete relaxation of the twitch was assessed, as described in Results. Tctonic re.pori.se.c were elicited by trains of pulses ranging in frequency from 50 to ROO Hz.The f i r i o n ,freyuanrj.of the tetanic stimulation was determined, i.e. the lowest stimulus frequency to obtain a totall) fused tension output with the present recording system. The minimum level of detection of the \ensiti\e tension delice was just below 10 mg, which was sufficient for the determination of fusion frequency even i n the newborn muscles, with tetanus tension of 1-3 g. The rria.uir?iunr tetanic ler7siun and the r-circ of rtwsion rise was measured for different stimulus frequencies. The rate of tension rise was taken as the steepest slope of the rising part of tetanic response. The values were expressed as the tension change in (Sch) was injected i.v. in a dose percent of the maximum tetanic tension per millisecond. S~cccin~~lrholirie of 1 mg per kg b.w., and the contracture induced in the inferior oblique by this drug was recorded. The animals were ventilated on pump during the transient paralysis of the respiratory muscles. Srtrrisrical analysis. When both muscles in the same animal were used, the values were lumped. Analysis of variance (one way) was performed for each parameter studied in order to reveal significant differences between any of the age groups. For a more detailed comparison between age groups, confidence intervals were calculated according to the method of multiple comparisons of Scheffe (1961). In all graphic presentations of results mean values have been plotted. The standard deviations for each parameter were of the same order of magnitude in all age groups, and could therefore be represented by the over all standard deviation from the analysis of Lariance. This is given in the legend to each diagram.
Results Twitch mid tetariic temiou. Values for maximum twitch and tetanus tension produced by the inferior oblique muscle at various ages are given in Fig. 1 A and B, respectively. Twitch and
135
TWITCH AND TETANUS IN KITTEN EYE MUSCLE
//
5g 1.0
L
0.5
I-
A
Fig. 1 . Changes with age in twitch tension (A), tetanic tension ( B ) and twitchitetanus ratio (C). Average values are presented for each age group. The overall standard deviation calculated in analysis of variance (see Methods) is in A: k0.29 g, B: 1 2 . 0 2 g, and
tetanus tension increased with age in about the same manner. At all ages the maximum tetanic tension was obtained at stimulus rates that produced a smooth tension curve, i.e. at the fusion frequency. This is well known from previous studies of adult cat eye muscle (Barmack, Bell and Rance 1971, Lennerstrand 1974), and seems to hold also for developing eye muscles. Twitchltetanrts ratio. The mean twitchitetanus ratios for each age group are shown in Fig. 1 C . The values were low at birth. They shifted to a higher level in animals between 2 and 10 weeks of age but became low again in the adult cat. These age variations were staristically significant at the 5 % level. Twitch co/~tractiorr.Contraction times ( c t ) and half-relaxation times (hrt) of the twitch have been plotted against age in Fig. 2 A and B. For comparison, mean values of the same parameters during development of the gastrocnemius and soleus muscles, obtained from the work by Hammarberg and Kellerth (1975 a), have been included in the graphs. Disregarding the fact that the ct and hrt values were much lower for the inferior oblique muscle than for the hindlimb muscles, it was found that the postnatal changes in ct and hrt were qualitatively similar in the inferior oblique and the fast gastrocnemus muscles, except at the ages above 10 weeks (Fig. 2 A, B). The cf and hrt at birth were approximately twice as large as the values obtained in the adult animal. In the inferior oblique the ct and the hrt continued to decrease beyond 20 weeks of age while the development with respect to speed of twitch contraction was completed within 6 weeks of age in the gastrocnemius muscles. As seen in Fig. 2 A the recution in ct of the inferior oblique muscle was more or less h e a r with age. The decline of the hrt (Fig. 2 B) was slower in the age period between 6 and 20
136
2'
GUNNAR LENNERSTRAND A N D JERKER HANSON A 8
=m
I
0,
5 !
2
to
5
Fig. 2. Twitch contraction time ( [ I ) in A and half-relaxation time (hrr) in B plotted against age for the inferior oblique (lo),the gastrocnemius ( G ) and soleus ( S ) muscles. The values of G and S obtained from the work by Hanimarberg and Kellerth (1975 a ) (courtesy of Dr J.-0. Kellerth). Note, that the left ordinate scale pertains to 10 readings and the right scale to G and S values.
week3 than at earlier and later ages, but the difference in ct and hrt development was not statistically significant. This was tested by calculating the cflhrt ratio for each muscle and comparing the values of the different age groups. Twitch drrration. In Fig. 3 A, showing twitch responses from kitten eye muscles of age 0 and 6 weeks and from an adult muscle, it can be seen that total twitch duration was shorter in the youngest than in the older muscles. Since the hrt was longer the younger the muscle (Fig. 2 B), it must be the latest phase of the twitch that tailed off more slowly in the older
A 0 weeks
6 weeks
4
Adult
li 0 1 0 1 2
4
6
10
__ 20
three different inferior oblique muscles of ages marked. Broken Fie. 3 line indicates resting'tension before stimulation. The decline of the twitch response is of longer duration in the 6 weeks old animal and the adult cat than in the new-born animal. Bars to the right of traces indicate i n top panel 0.2 g, and in middle and bottom panels 0.5 g. Same time scale in all recordings. B. Twitch duration estimated from the lowest stimulus frequency to produce summation between the twitches in double stimulation. Note, that stimulus frequency was higher and twitch duration thus shorter in young than i n old kittens. Average values given for each age group. S.D.: k0.9 Hz.
137
TWITCH AND TETANUS IN KITTEN EYE MUSCLE
tgv * b = 64.14
b
0
15
b = -163.3
c
E 80
. 0
6-
0
-100 ’
0.20
0.30 0.40 stim. strength ( V )
0 1 2
4
7
6 10 age (weeks)
/+-it--
20
adult
Fig. 4.Changes in ct and hrt with increase in intensity of nerve stimulation. A shows the initial increase in ct with stimulus intensity in a muscle from a newborn kitten and B the decrease in ct in the case of a 20 week old animal. At supramaximal stimulus intensities ct levelled off. The parameters a, b and c from the equation in the text are marked. These values and the regression lines we calculated on a computor using the “metod of least squares”. In C mean slope values (6) for relations between stimulus intensity and cr or hrt have heen plotted for all age groupes. The overall standard deviation was & 44.9 for ct and & 103.8 for Art. The graph indicates that young muscles have a positive slope and older muscles a negative slope. This difference between age groups was statistically significant only for ct.
muscles. The time for total twitch tension decline was estimated from double twitch responses to stimulation at low repetition rates (10 Hz and below; see also Hanson and Lennerstrand 1977). At very low stimulus frequencies the first twitch had completely vanished before the response to the second stimulus appeared. The lowest frequency at which the two twitches started to show summation has been plotted in Fig. 3 B for muscles of different ages. The values for the newborn cat were the highest. Thus, in spite of the long hrt at this age, the decline of the twitch response was faster than in older muscles. The difference between newborn and 6 weeks old animals was statistically significant (p < 0.01). Twitch contraction and stimulus strength. On increasing the intensity of nerve stimulation from threshold to supramaximal values, more and more motor units were recruited in the muscle, which lead to an increase in twitch amplitude and to changes in ct and k t . The latter were studied in some detail, since it has been shown previously that ct and hrt of adult cat eye muscle, in contrast to other skeletal muscle, had higher values at threshold than at maximal stimulus intensities (Lennerstrand and Bach-y-Rita 1974). This means that ct and hrf decreased with increasing stimulus strength in the lowest intensity range. Later, as the maximal response level was approached, the twitch ct and hrt increased with stimulus intensity. The youngest muscles, on the other hand, showed a steady increase in both ct and
I38 TABLE
-
GUNNAR LENNERSTRAND AND JERKER HANSON
I I . Twitch c f and hrt. obtained with threshold and supramaximal stimulation, respectively, in kitten eye muscle of different age. Mean values ( k S . D . ) are given.
Age
llrf
tf
Threshold week weeh neck\ \*eeh\ 6 weel\ 10 weehs 20 w e e h \ 0 I 2 4
Adult
Supramax.
Threshold
Supramax.
15.3 ( 2 2.9) 14.4 ( k O . 8 ) 13.1 ( 1 0 . 8 ) 12.0 ( 2 0 . 9 ) 10.0 ( k 0 . 8 ) 9.0 (11.5) 7.0 ($0.21) 6.0 (1 0.8)
13.5 ( 2 5 . 7 ) 13.1 (-+ 1.5) 11.4 ( 2 1.8) 12.0 ( i 2 . 5 ) 11.6(C2.0)
16.0 ( ~ 3 . 0 ) 18.8 ( 1 4 . 8 ) 14.8 ( ? 2.21 13.0 (21.5)
13.3 ( - 3 . 7 ) 15.3 ( 57.0) 11.0 ( 51.4)
11.0 (F1.5) 10.8 (k2.1) 7.4 (+- 1.6)
12.0(f1.8)
hrt M i t h increasing stimulus strength as exemplified in Fig. 4 A for twitch c f . A change into the adult pattern was observed in kittens 10-70 weeks of age (Fig. 4 B). 111order to compare twitch parameters in musrles of different age. the relation between the stimulus intemit! and c f or lirt was approximated from the graphs as shown in Fig. 4 and expressed in following may
where r is threshold intensity. c intensity of reach maximal response level x . and 6 the slope of the curve (see Fig. 4 A). A positive slope denotes an increase in cI or hrr with stimulus strength a n d a negative slope the reterse. An analysis of \ariance a n d a calculation of confidence intervals according to the method of Scheff+ (1961) was performed for the 6-values of muscles from cats of different age.
Significant differences were revealed for ct of kittens below ten weeks of age and older cats. However, there were no statistical difference between any age groups with respect to krt changes with stimulus intensity, although the mean values of ct and hrt seem to vary in a similar way with age (Fig. 4 C). The standard deviation for hrt was much larger than for ct. These findings strongly support the idea that the recruitment of motor units to increasing stimulus intensity followed different patterns in young and in adult eye muscles, with a period of transition at 6-10 weeks.
2001
f -
0 1 2
4
6
10
_
c
dlh
Agclmsktl
Fig 5 Changes in fubioii frequency of tetanic contraction with age. Average values plotted; standard dekiation: 2 2 7 5 H7.
TWITCH AND TETANUS I N KITTEN EYE MUSCLE
Fig. 6. Maximal rate of tension rise in tetanic contractions for different age groups. For comparison between age groups, the rate of tension rise for each muscle has been related to the maximal tetanic terision of that muscle (Po)and expressed in percent of P, per msec. Standard deviation & 0.8";.
139
21
Absolute values for ct and hrt at threshold and at maximal activation are given in Table 11. It was found that the threshold C I , but not hrf increased continuously with age. Fusion frequency. As shown in Fig. 5 the fusion frequency was around 200 Hz in the newborn kitten and increased with age until the values saturated at about 325 Hz for muscles older than 6 weeks. The difference between values above 6 weeks of age and those below age 2 weeks was statistically significant (p 20.01). Maximal rate of tension rise. Stimulus frequencies higher than the fusion frequency had to be applied in all age groups in order to attain maximal rate of rise of the tetanic response. In the newborn animal stimulation at 400 Hz was necessary and in the adult cat 600 Hz. Mean values of rate of tension rise in Fig. 6 have been expressed as the tension increase in percent of maximal tetanic tension (Po) per millisecond. There was no significant variation between muscles up to 6 weeks of age. At 10 weeks the values started to increase and at 20 weeks they were even highei than in the adult animal. Effect o f snccinylcholine (Sch). This drug is known to cause a long-lasting contracture in extraocular muscle of the adult cat, probably by inducing an extensive depolarization of the TABLE 111. Effects of succinylcholine on inferior oblique muscles at different ages. Drug action has been expressed as succinylcholine-induced tension in percent of the maximal tetanic tension, and has been measured at the time of maximal succinylcholine effect and 5 and 10 min later. Mean values (& S.D.) are given for each age group. Age 0 week 1 week
2 weeks 4 weeks 6 weeks 10 weeks 20 weeks Adult
Max effect
5 min
10 min
140
GUNNAR LENNERSTRAND AND JERKER HANSON
multiply innervated fibres (Bach-y-Rita 1971). A contracture could be elicited by the drug in kitten inferior oblique muscle of all ages. Drug action was expressed as the Sch-induced tension in percent of the maximuin tetanic tension. There was no significant difference in drug action between age groups (Table 1"). The drug effects 5 and 10 min after the injection were slightly larger in the older animals. Duration of drug action was 10 to 15 min in animals at age 10 weeks and below, and slightly longer (15-20 min) in the older animals.
Discussion Postriutal clifferentiutiati of fast arid slow fibres
At birth the inferior oblique was a much slower muscle than in the adult cat. The speed of twitch contraction increased gradually over at least the first half year of the kitten's life. I n this respect the development of eye muscles was similar to that of fast limb muscles, although in the latter adult values were already obtained at the age of 2 months (Buller et a/. 1960, Buller and Lewis 1965, Mann and Salafsky 1970, Westerman et a/. 1973, Hammarberg and Kellerth 1975 a). However, if speed of contraction in eye muscles instead was estimated from the stimulus frequency to produce a fused response, the mature stage would seem to be reached by 6 weeks. By using the rate of tension rise as the measure of contraction speed, one would conclude that muscle development did not start until about 6 weeks of age and continued to approxiniately age 20 weeks. The differences between the postnatal development of fusion frequency and twitch contraction time might be related to post-tetanic effects. The twitch responses reported in this paper were not influenced by previous muscle activity. It will be shown in a subsequent paper (Lennerstrand and Hanson 1978) that repetitive stimulation prolonged twitch relaxation and induced a staircase phenomenon in the tetanic response. These effects were most marked in the older animals, and could account for most of the levelling off of the curve relating fusion frequency to age. In order to explain the developmental changes in the rate of tension rise of eye muscles one might propose that the slow fibres matured more rapidly than fast fibres. Constant values for the rate of tension rise during the first 6 weeks in spite of a decrease in twitch contraction time would indicate that the slow fibre system completed most of its development during this period. The parallel changes in twitch contraction time and rate of tension rise up to age 20 weeks, suggest that the fast fibres obtain their adult properties much later. Another finding to support the idea that the development of slow and fast fibres of eye muscles followed different time courses was that the time for total twitch relaxation increased from birth to six weeks of age, which would indicate that the slowest fibres, which contribute most to the latest phases of the twitch response, not only remained slow from birth but became still slower with age.
The e j u miscIe resporises to threshold neri>estimulation It was found that increasing the intensity of nerve stimulation from the threshold to the supramaximal level affected twitch responses differently depending on the age of the muscle. In the young muscles, fast fibres seemed to be activated first and as stimulus intensity rose,
TWITCH AND TETANUS IN KITTEN EYE MUSCLE
141
more and more slow fibres were recruited. The same pattern is seen in ordinary skeletal muscle irrespective of age. However, in cat eye muscle of age 10 weeks the response to threshold stimulation was slower than those to supra-threshold stimulus intensities. This pattern is regularly seen in the adult muscle (Lennerstrand and Bach-y-Rita 1974). The muscle fibres responding at threshold must be innervated by large diameter nerve fibres, since these would be activated by the lowest stimulus. It seems unlikely that the muscle fibres in question would be innervated by small nerve fibres in the young animal and later re-innervated by large nerve fibres, or that the nerve fibres to these muscle fibres should have grown at a faster rate than other nerve fibres. Fast hindlimb muscles are known to develop larger nerve fibres than slow muscle (Nystrom 1968 b), as reflected also in the higher conduction velocities of nerve axons innervating fast motor units than slow motor units (Bagust et a1. 1973, Bagust 1974). This relation holds also for kitten hindlimb muscle (Bagust, Lewis and Westerman 1974). Since the number of muscle fibres seems to remain unchanged during postnatal development (Westerman et al. 1973), the most plausible explanation for the appearance in eye muscles of a slow response to threshold nerve stimulation at 10 weeks of age would seem to be that some portion of the muscle fibres had changed their twitch properties and developed slightly slower contractions, in the same manner as the slow fibres in the soleus muscle (cf. Fig. 2 A and B). These fibres may be located in the outer, orbital layer, since findings in the adult cat indicate that the motor response to threshold stimulation arises in orbital fibres (Lennerstrand and Bach-y-Rita 1974, Bach-y-Rita 1975). The histochemical study of postnatal changes in eye muscles (Hanson et al. 1977) has shown that the orbital layer is poorly defined at birth but fully developed at about 10 weeks of age.
Effects of sirccinylcholine The succinylcholine experiments gave similar results at all stages of development. In response to an injection of the same amount of the substance per unit b.wt., the muscles developed contractures that amounted to 40-50% of the maximal tetanic tension. The drug is thought to induce a long-lasting contraction of multiply innervated muscle fibres, i.e. the slow fibre components, by depolarizing the postsynaptic region of the muscle fibre membrane (Bach-yRita 1971). In eye muscle of the adult cat the drug would seem to act on multiply innervated fibres of both the amphibian and the avian types (Bach-y-Rita P t al. 1977). The multiple innervation of adult extraocular muscle is probably only to a very small extent of the polyneuronal type, with overlapping innervation from several nerve fibres to the same muscle fibre (Bach-y-Rita and Lennerstrand 1974). It is well-known, however, that all fibres in kitten hindlimb muscle are polyneuronally innervated at birth, but that this type of innervation has disappeared at 6 weeks of age (Bagust, Lewis and Westerman 1973). The possibility of polyneuronal innervation of eye muscle fibres in young kittens makes it difficult to draw firm conclusions on the development of multiply innervated fibres on the basis of the succinylcholine expts. We wish to thank Stig Danielsson for advice and assistance in the statistical analysis. This study was supported by grants from Karolinska institutets fonder and the Swedish Medical Research Council (grants No. 4751,4719 and 3875).
142
GUNNAR LENNERSTRAND AND JERKER HANSON
References A . a n d C. \ A X HORN.Muscle cell types of the cat inferior oblique. In: Bnsic mcdianisnis of ocular rriorilirr urttl their c/iniccrl inrplications. Eds. G . Lennerstrand a n d P. Bach-y-Rita. Pergamon Press, Oxford, 1975. pp. 15-43, BACH-Y-RITA, P.. Neurophqsiology of eye movements. In: The control of e.re niowriient.s. Eds. P. Bach-yRita. C. C . Colins a n d J . E. Hyde, Academic Press, New York. 1971. pp. 7-45. BACH-Y-RITA, P.. StrLictural-functional correlations in eye muscle fibers. Eye muscle proprioception. In: Bn\ic nrerliaiiitms of iiculor mofi/irJ.nnd rheir clinical iniplicalions. Eds. G . Lennerstrand a n d P. Bach-yRita. Pergamon Press. Oxford. 1975. pp. 91-109. BACH-Y-RITA, P. and F. ITO, In vivo studies on fast and slow muscle fibers in cat extraocular muscles. J. y r n . P l r ~ ~ ~ i1966. o l . 49. 1177-1 198. BATH-Y-RITA. P. and G . LENNERSTRANU, Absence of polyneuronal innervation in cat extraocular muscles. J . P / i , ~ t i o /(. L o n d . ) 1975. 244. 613-624. BAC-H-Y-RITA. P.. G. LENNERSTKAND, J. ALVAKAUO, K . NICHOLS a n d G . MCHOLM,Extraocular muscle fibers: ultrastructural identification of iontophoreticall\i labeled fibers contracting in response t o succin) lcholine. / n r r s / . Ophtlinl Visual Sci. 1977. 161. 561-565. BAOL-$1,J . . Relationships between motor newe conduction velocities a n d motor unit contraction characteri\tics in a slow twitch muscle of the cat. J . Ph.uiol. (Lond.) 1974. 238. 269-278. Isometric contractions of motor B A O L ~ J., T , S . KKOTT. D. M. LEWIS,J. C. L U C Kand R. A. WESTERMAN, units in a fast twitch muscle of the cat. J . PIi.r~iol.(Lond.) 1973. 231. 87-104. BALL:ST.J . . D. M . Ltwis and R . A . WESTERMAW, Polyneuronal innervation of kitten skeletal muscle. J. P / i j s i o l . (Lond.) 1973. 22Y. 241-255. BAGVST,J . . D. M . Ltwis and R . A. WESTERMAN, T h e properties of motor units in a fast and a slow twitch /. 1974. 237. 75-90. muscle during post-natal development in the kitten. J . P h , ~ ~ i o(Lond.) BARMACK,N. H . , C. C. BELLa n d B . C . RENCE,Tension and rate of tension development during isometric rehponses of estraocular niuscls. J . h'curop/iuiu/. 1971. 34. 1072-1079. BiTt.tR, A. J. and D. M. LEWIS. Further obserbations on the differentiation of skeletal muscles in the kitten hind limb. J . P / i I . s i o / . (Lond.) 1965. 176. 355-370. BCi.i.tK, A. J . , J. C. E C C L ~and S R . M. ECCLES,Differentiation of fast a n d slow muscles in the cat hind limb. J . Phj \ i d . ( L o n d ) 1960. 150. 3 9 9 4 1 6 . CLOSF. R. I., D) naniic properties of mammalian skeletal muscles. Ph.vsio/. Rerieivx. 1972. 52. 129-197. CLfm. R . I . , Specialization among fast-twitch muscles. In: Exploratory concepts in muscular dystrophy. Ed. A. T. Milhorat. E\-cerpta M e t l . int. Congr. Srr. N o . 333. 1975. pp. 309-318. CLOSF,R . and J . F. Y . HOH. Force: velocity properties of kitten muscles. J . P/i.rsiol. 1967. 192. 815-822. H A M ~ ~ ~ R B C.. E K The C , hictochemical appearance of developing muscle fibres in the gastrocnemius, soleus and anterior tibia1 muscles of the kitten, as \iewed in serial sections stained for lipids and succinic dehydrogenase. ,4cta nrurol. .scoricl. 1974. 50. 285--301. HAMMAIIBEHC, C . and J . - 0 . KtLLERTH. T h e postnatal development of some twitch a n d fatigue properties of the ankle flexor and extensor muscles of the cat. Arra ph.vsiol. sraml. 1975 a. 95. 166-178. T h e postnatal development of some twitch a n d fatigue properties HAhlhl ARIIEKG, C. and J.-0. KELLEKTH, of sinylc motor units in the ankle muscles of the hitten. Arta plij.sio/. srcmd. 1975 b. Y5. 243-257. HANSOU, J . and G. L t h K E K S T R A K D , Contractile and histochemical properties of the inferior oblique muscle i n the rat and i n the cat. At,ra ophrol. (Kbh.) 1977. 5 5 . 88-102. HAYSON, J . . G . L E K ~ E R S T R and A W K . NICHOLS, The postnatal development of cat extraocular muscle. 11. Variations with age in appearance of fatigue and potentiation on repetitive stimulation. Electroen. C l h . Nrurophj.sio1. 1977. 42. 130. HAkSOK. J., K . NICHOLS and G . LENNERSTKANU. The postnatal development of the inferior oblique muscle of the cat. 111. Fiber sizes and histochemical properties. (In preparation.) HESS. A. a n d G . PILAR, Slow fibres in the extraocular muscles of the cat. J . Ph.~~sicd.(Lond.) 1963. 169. 780-797. L ~ I W K E K S T R AG.. N DElectrical , activity and isometric tension in motor units of the cat's inferior oblique m u x l e . Ac.to pli.uiol. .wand. 1974. 51. 458-474. ~ R S ~ R A N D G.. . Motor units in eye muscles. In: Basic rrieclirrnisms of ocular n~orilityand rhcir clinical iniplrcotionv. Eds. G. Lennerstrand and P. Bach-y-Rita. Pergamon Press, Oxford. 1975. pp. 119-143. II-~~ERSTKA G.NaDn d, P. BACH-Y-RITA, Activation of slow motor units by threshold stimulation of cat r ) e niu.icle nerves. l n w s r . ophtlial. 1974. 13. 879-882. . ~ L V A K A D V . J.
TWITCH AND TETANUS IN KITTEN EYE MUSCLE
I43
LENNERSTRAND, G. and J. HANSON, The postnatal development of the inferior oblique muscle of the cat. 11. Effects of repetitive stimulation on isometric tension responses. Arfaphysiol.srand. 1978.103. 144-153. LENNERSTRAND, G., J. HANSON and K. NICHOLS, The postnatal development of cat extraocular muscle. I. Variations with age in speed of muscle contraction. Electroen. Clin. Neurophysiol. 1977. 42. 129. MAIER,A. and E. ELDRED, Postnatal growth of the extra- and intrafusal fibers in the soleus and medial gastronemius muscles of the cat. Anier. J. Anat. 1974. 141. 161-177. MANN,W. S. and B. SALAFSKY, Enzymic and physiological studies on normal and disused developing fast and slow cat muscles. J. Physiol. (Lond.) 1970. 208. 33-47. MATYUSHKIN, D. P., Contractions and their correlations with the action potentials in the phasic fibres of the extrinsic eye muscles of adult and newborn animals. Biophysics 1967. 12. 528-536. MAYR,R., Discussion of the paper by Alvarado and van Horn. In: Basic nierhanisms of ocular nmti1it.y and their clinical implirulions. Eds. G . Lennerstrand and P. Bach-y-Rita. Pergamon Press. Oxford. 1975. pp. 44-45. NYSTROM, B.. Mechanical and electrical responses to single shocks in developing cat leg muscles following tetanization. Arta physiol. srand. 1968 a. 74. 207-225. B., Histochemistry of developing cat muscles. Arta neurol. scanrl. 1968 b. 44. 4 0 5 4 3 9 . NYSTROM, PEACHEY, L., The structure of the extraocular muscle fibers of mammals. In; The rontrol q f e j e mownients. Eds. P. Bach-y-Rita, C. C. Collins and J. E. Hyde. Academic Press, New York. 1971. pp. 47-66. PEACHEY, L., M. TAKEICHI and A. C. NAG, Muscle fiber types and innervation in adult cat extraocular muscles. In: Exploratory concepts in muscular dystrophy. Ed. A. T. Milhorat. Excerpfa Med. int. Congr. Ser. No. 333. 1975. pp. 246-257. SCHEFFB,H., The una\.vsis of uariance. 2nd edition. John Wiley and Sons, London. 1961. WESTERMAN, R. A., D. M. LEWIS,J. ~ A G U S T ,G. D. EDJTEHADI and D. PALLOT,Communication between nerves and muscles: postnatal development in kitten hind limb fast and slow twitch muscle. In: Memory and tran$er of information. Ed. H. P.Zippel. Plenum Press, New York. 1973. pp. 255-291.
The postnatal development of the inferior oblique muscle of the cat I. Isometric twitch and tetanic properties BY GUNNAR
LENNERSTRAND and JERKER HANSON Received 28 November 1977
Abstract LENNERSTRAND, G. and HANSON, J. The postriatal dewlopmerit of the inferior oblique muscle of the cLit. I. Isometric fwilch arid tetnriic properties. Acta physiol. scand. 1978. 103. 132-143. The posrnatal development of the inferior oblique muscle in the cat has been studied with physiological and hictochemical tecknique5. This paper describes the changes with age in isometric twitch and tetanic reymnse characteristics. The twitch amplitude increased and the twitch contraction time (cr) and halfrelaxation time (hrr) decreased almost linearly from birth to adulthood. The relation between the strength of n e n e stimulation and twitch cr and / t r / changed during development with a threshold slow response appearing at 10 weeks. Twitch responses in cats 6 weeks of age or older were of longer duration than in younger c a ts in spite of the longer c f and hrf in young cats. Fusion frequency of the tetanic response reached a constant level in muscles 6 weeks or older. The maximum rate of tension rise remained the same from birth to 6 ueeks of age and later increased markedly up to 20 weeks of age. The contracture induced by succin\Icholinc was the same in muscles of all ages. These data were related to previous findings on the postnatal development of fast and slow muscles and motor units in the hindlimb of the cat. A differentiation of the de\elopment of fast and slow eye muscle fibrcs is suggested. Slow fibres seemed to have completed their niaturation a t about ten wceks of age, while the development of fast fibre properties continued, probably u p ro the adult stage. Kc?, wordv: Postnatal de\elopment, cat extraocular muscle, fast and slow fibres, contractile properties, twitch aiid tetanus
structure, adult e y e muscle is known t o differ markedly from other muscles. The inferior oblique muscle of the cat has been shown to contain si n g l y (focally) innervated muscle fibres of t h e same t y p e s t h a t exist in mammalian skeletal muscles (Alvarado and van Horn 1975), but in addition m u lti p l y innervated fibres, not seen in ordinary skeletal muscle, h a v e been identified (Hess and Pilar 1963, Peachey, Takeichi and Nag 1975, Alvarado and van Horn 1975, Mayr 1975). The multiply innervated fibres are of 2 types, one of t h e m resembling slow fibres in amphibian muscle and t h e o t h e r slow f ibr e s in avian muscle (Peachey 1971, Peachey et a!. 1975, Alvarado and van Horn 1975, M a y r 1975). W i t h r e g a r d t o muscle fibre
132
TWITCH AND TETANUS IN KITTEN EYE MUSCLE
I33
Three types of motor units with different functional properties have been identified in the cat inferior oblique muscle (Lennerstrand 1974, 1975). The units with the fastest contractions are probably composed of singly innervated fibres and the slowest units of multiply innervated fibres of the slow amphibian type. The units of intermediate speed of contraction have been suggested to consist of multiply innervated fibres resembling the slow fibres in avian muscle (Bach-y-Rita and Ito 1966, Lennerstrand 1974, 1975, Lennerstrand and Bach-yRita 1974, Bach-y-Rita 1975). It is not known how the properties of eye muscle fibres in the cat change from birth to the adult stage. Matyushkin (1967) reported that the contraction of eye muscles in young rabbits were slower than those of adult muscle, and similar observations have been made on rat eye muscle by Close (1975). In recent years the postnatal development of skeletal muscle in the cat has been extensively studied, particularly the functional differentiation of the fast and slow twitch fibre systems in hindlimb muscles (Buller, Eccles and Eccles 1960, Buller and Lewis 1965, Close and Hoh 1967, Mann and Salatsky 1970, Bagust, Lewis and Westerman 1973, Westerman et al. 1973, Hammarberg and Kellerth 1975 a, b). At birth all hindlimb muscles contract slowly. They seem to be composed of the same types of fibres (Nystrom 1968 b, Maier and Eldred 1974, Hammarberg 1974). During the first postnatal weeks a differentiation takes place, and some muscles like the gastrocnemius increase their speed of contraction with age, while others like the soleus remain slow throughout development (Buller et al. 1960, Buller and Lewis 1965, Nystrom 1968 a, Westerman et al. 1973). The differentiation of fast and slow contraction seems to occur in parallel with changes in fibre composition (Nystrom 1968 b, Maier and Eldred 1974, Hammarberg 1974). The purpose of the present investigation has been to study the postnatal development of cat extraocular muscle with physiological and histochemical techniques. A comparison with the development of fast and slow fibre systems of other cat muscles will be attempted although this will be somewhat difficult due to the lack of information on the postnatal development of muscle fibres with multiple innervation, even in amphibian and avian muscle. Isometric contractions have been recorded in the inferior oblique muscle of kittens of different age and in the adult cat. It has not been possible to record from individual fibres or motor units, and conclusions have to be drawn from data obtained from the whole muscle. In this paper the postnatal changes in twitch and tetanic conctraction will be evaluated. In a subsequent paper the changes with age in fatigue properties and other post-tetanic effects will be examined (Lennerstrand and Hanson 1978). The developmental changes in the histochemical fibre spectrum of the inferior oblique muscle will be described separately (Hanson, Nichols and Lennerstrand 1978). Preliminary reports on the results have been published elsewhere (Hanson, Lennerstrand and Nichols 1977, Lennerstrand, Hanson and Nichols 1977).
Methods 25 kittens and 3 adult cats were used in the study. They were arranged in 8 groups according to postnatal age. The number of animals in each group and their body weight are shown in Table I. In most animals muscles of both eyes were used for experiments. The animals were anesthetized with pentobarbital (NembutaP) 40 mg/kg b.w. injected iritraperjtoneally and the trachea and one femoral vein were cannulated. Additional doses of anesthetic were given i.v. when needed. The head was rigidly clamped with earpins and a bite bar. The inferior oblique, the lateral and
I34
GUNNAR LENNERSTRAND AND JERKER HANSON
T A M E1. Presentation of the groups of experimental animals in the study. Group 0 weeh 1 weeh
Age (days) 2-3 6-9
2 week\
12-17
4 weeks
29-32
6 \reel\,
4245 66-74 123. 1 5 1 over 365
10 v,eeks
20 Heehs Adult
Weight (g)
Animals
Muscles
138-143 195-26 1 22 1-326 308487 348-550 600-900 650. I 200 2 800-3 500
medical recti and the retractor bulbi muscles were freed from the globe, which was enucleated. The nictitating membrane was raised to form a pool in the orbit, which was filled with warm mineral oil (35-37°C). The bod! temperature was monitored continuously and regulated to 37-38'C with a beating pad. After the experiments parts of the muscles were removed for histochemistry. /.\or?ietrir rcnsroti recording. The short tendon of the inferior oblique muscle was tied with braided sutures to a hook glued to one end of a sensitive strain gauge (Endevco no. 8101). The other end of the strain gauge was cldniped to a metal base attached to a micromanipulator. The undamped reasonance frequency in air of the tension recording assembly was 2 kHz. The linear range of the system spanned froni 10 mg to 40 g. The signals from the strain gauge \\ere amplified and displayed o n a Medelec UV recording unit. The passhand of the amplifiers was set at 0 4 0 kHz. In order to enable comparison between contractions in different muscles, a11 tension recordings were made at the muscle length of maximal twitch response. Care was taken to align the strain gauge perpendicular to the natural pulling direction of the muscle. Stitiiihriorr. The muscle nerve was carefully dissected free from orbital tissue and cut as far centrally 3 s possible. I t w a s positioned over paired platinum electrodes and stimulated with square-wave pulses of 0.1 n i \ duration. generated by a Grass S8 stimulator and fed through an isolation unit. R ~ o r d r r i parid r ~ i ~ ~ u . ~ i ~ r ~ With ~iii~~ some r i t . \ ,slight modifications the techniques have been similar to those used by Haiison and Lennerstrand (1977). Twirclr rrspumcs were recorded first to increasing intensities of single pulse stimulation from threshold to supramaximal values. Threshold was about 0.4 V for the newborn animals and decreased to about 0.2 V for adult cats. Stimulus intensities at least five times those for rnawnial raitch response were used for the res: of the recordings of twitch and tetanic responses. The maximal amplitude of the twitch, the contraction time ( r t ) and the half-relaxation time (hrt) of the twitch contraction were measured. I n addition the time for complete relaxation of the twitch was assessed, as described in Results. Tctonic re.pori.se.c were elicited by trains of pulses ranging in frequency from 50 to ROO Hz.The f i r i o n ,freyuanrj.of the tetanic stimulation was determined, i.e. the lowest stimulus frequency to obtain a totall) fused tension output with the present recording system. The minimum level of detection of the \ensiti\e tension delice was just below 10 mg, which was sufficient for the determination of fusion frequency even i n the newborn muscles, with tetanus tension of 1-3 g. The rria.uir?iunr tetanic ler7siun and the r-circ of rtwsion rise was measured for different stimulus frequencies. The rate of tension rise was taken as the steepest slope of the rising part of tetanic response. The values were expressed as the tension change in (Sch) was injected i.v. in a dose percent of the maximum tetanic tension per millisecond. S~cccin~~lrholirie of 1 mg per kg b.w., and the contracture induced in the inferior oblique by this drug was recorded. The animals were ventilated on pump during the transient paralysis of the respiratory muscles. Srtrrisrical analysis. When both muscles in the same animal were used, the values were lumped. Analysis of variance (one way) was performed for each parameter studied in order to reveal significant differences between any of the age groups. For a more detailed comparison between age groups, confidence intervals were calculated according to the method of multiple comparisons of Scheffe (1961). In all graphic presentations of results mean values have been plotted. The standard deviations for each parameter were of the same order of magnitude in all age groups, and could therefore be represented by the over all standard deviation from the analysis of Lariance. This is given in the legend to each diagram.
Results Twitch mid tetariic temiou. Values for maximum twitch and tetanus tension produced by the inferior oblique muscle at various ages are given in Fig. 1 A and B, respectively. Twitch and
135
TWITCH AND TETANUS IN KITTEN EYE MUSCLE
//
5g 1.0
L
0.5
I-
A
Fig. 1 . Changes with age in twitch tension (A), tetanic tension ( B ) and twitchitetanus ratio (C). Average values are presented for each age group. The overall standard deviation calculated in analysis of variance (see Methods) is in A: k0.29 g, B: 1 2 . 0 2 g, and
tetanus tension increased with age in about the same manner. At all ages the maximum tetanic tension was obtained at stimulus rates that produced a smooth tension curve, i.e. at the fusion frequency. This is well known from previous studies of adult cat eye muscle (Barmack, Bell and Rance 1971, Lennerstrand 1974), and seems to hold also for developing eye muscles. Twitchltetanrts ratio. The mean twitchitetanus ratios for each age group are shown in Fig. 1 C . The values were low at birth. They shifted to a higher level in animals between 2 and 10 weeks of age but became low again in the adult cat. These age variations were staristically significant at the 5 % level. Twitch co/~tractiorr.Contraction times ( c t ) and half-relaxation times (hrt) of the twitch have been plotted against age in Fig. 2 A and B. For comparison, mean values of the same parameters during development of the gastrocnemius and soleus muscles, obtained from the work by Hammarberg and Kellerth (1975 a), have been included in the graphs. Disregarding the fact that the ct and hrt values were much lower for the inferior oblique muscle than for the hindlimb muscles, it was found that the postnatal changes in ct and hrt were qualitatively similar in the inferior oblique and the fast gastrocnemus muscles, except at the ages above 10 weeks (Fig. 2 A, B). The cf and hrt at birth were approximately twice as large as the values obtained in the adult animal. In the inferior oblique the ct and the hrt continued to decrease beyond 20 weeks of age while the development with respect to speed of twitch contraction was completed within 6 weeks of age in the gastrocnemius muscles. As seen in Fig. 2 A the recution in ct of the inferior oblique muscle was more or less h e a r with age. The decline of the hrt (Fig. 2 B) was slower in the age period between 6 and 20
136
2'
GUNNAR LENNERSTRAND A N D JERKER HANSON A 8
=m
I
0,
5 !
2
to
5
Fig. 2. Twitch contraction time ( [ I ) in A and half-relaxation time (hrr) in B plotted against age for the inferior oblique (lo),the gastrocnemius ( G ) and soleus ( S ) muscles. The values of G and S obtained from the work by Hanimarberg and Kellerth (1975 a ) (courtesy of Dr J.-0. Kellerth). Note, that the left ordinate scale pertains to 10 readings and the right scale to G and S values.
week3 than at earlier and later ages, but the difference in ct and hrt development was not statistically significant. This was tested by calculating the cflhrt ratio for each muscle and comparing the values of the different age groups. Twitch drrration. In Fig. 3 A, showing twitch responses from kitten eye muscles of age 0 and 6 weeks and from an adult muscle, it can be seen that total twitch duration was shorter in the youngest than in the older muscles. Since the hrt was longer the younger the muscle (Fig. 2 B), it must be the latest phase of the twitch that tailed off more slowly in the older
A 0 weeks
6 weeks
4
Adult
li 0 1 0 1 2
4
6
10
__ 20
three different inferior oblique muscles of ages marked. Broken Fie. 3 line indicates resting'tension before stimulation. The decline of the twitch response is of longer duration in the 6 weeks old animal and the adult cat than in the new-born animal. Bars to the right of traces indicate i n top panel 0.2 g, and in middle and bottom panels 0.5 g. Same time scale in all recordings. B. Twitch duration estimated from the lowest stimulus frequency to produce summation between the twitches in double stimulation. Note, that stimulus frequency was higher and twitch duration thus shorter in young than i n old kittens. Average values given for each age group. S.D.: k0.9 Hz.
137
TWITCH AND TETANUS IN KITTEN EYE MUSCLE
tgv * b = 64.14
b
0
15
b = -163.3
c
E 80
. 0
6-
0
-100 ’
0.20
0.30 0.40 stim. strength ( V )
0 1 2
4
7
6 10 age (weeks)
/+-it--
20
adult
Fig. 4.Changes in ct and hrt with increase in intensity of nerve stimulation. A shows the initial increase in ct with stimulus intensity in a muscle from a newborn kitten and B the decrease in ct in the case of a 20 week old animal. At supramaximal stimulus intensities ct levelled off. The parameters a, b and c from the equation in the text are marked. These values and the regression lines we calculated on a computor using the “metod of least squares”. In C mean slope values (6) for relations between stimulus intensity and cr or hrt have heen plotted for all age groupes. The overall standard deviation was & 44.9 for ct and & 103.8 for Art. The graph indicates that young muscles have a positive slope and older muscles a negative slope. This difference between age groups was statistically significant only for ct.
muscles. The time for total twitch tension decline was estimated from double twitch responses to stimulation at low repetition rates (10 Hz and below; see also Hanson and Lennerstrand 1977). At very low stimulus frequencies the first twitch had completely vanished before the response to the second stimulus appeared. The lowest frequency at which the two twitches started to show summation has been plotted in Fig. 3 B for muscles of different ages. The values for the newborn cat were the highest. Thus, in spite of the long hrt at this age, the decline of the twitch response was faster than in older muscles. The difference between newborn and 6 weeks old animals was statistically significant (p < 0.01). Twitch contraction and stimulus strength. On increasing the intensity of nerve stimulation from threshold to supramaximal values, more and more motor units were recruited in the muscle, which lead to an increase in twitch amplitude and to changes in ct and k t . The latter were studied in some detail, since it has been shown previously that ct and hrt of adult cat eye muscle, in contrast to other skeletal muscle, had higher values at threshold than at maximal stimulus intensities (Lennerstrand and Bach-y-Rita 1974). This means that ct and hrf decreased with increasing stimulus strength in the lowest intensity range. Later, as the maximal response level was approached, the twitch ct and hrt increased with stimulus intensity. The youngest muscles, on the other hand, showed a steady increase in both ct and
I38 TABLE
-
GUNNAR LENNERSTRAND AND JERKER HANSON
I I . Twitch c f and hrt. obtained with threshold and supramaximal stimulation, respectively, in kitten eye muscle of different age. Mean values ( k S . D . ) are given.
Age
llrf
tf
Threshold week weeh neck\ \*eeh\ 6 weel\ 10 weehs 20 w e e h \ 0 I 2 4
Adult
Supramax.
Threshold
Supramax.
15.3 ( 2 2.9) 14.4 ( k O . 8 ) 13.1 ( 1 0 . 8 ) 12.0 ( 2 0 . 9 ) 10.0 ( k 0 . 8 ) 9.0 (11.5) 7.0 ($0.21) 6.0 (1 0.8)
13.5 ( 2 5 . 7 ) 13.1 (-+ 1.5) 11.4 ( 2 1.8) 12.0 ( i 2 . 5 ) 11.6(C2.0)
16.0 ( ~ 3 . 0 ) 18.8 ( 1 4 . 8 ) 14.8 ( ? 2.21 13.0 (21.5)
13.3 ( - 3 . 7 ) 15.3 ( 57.0) 11.0 ( 51.4)
11.0 (F1.5) 10.8 (k2.1) 7.4 (+- 1.6)
12.0(f1.8)
hrt M i t h increasing stimulus strength as exemplified in Fig. 4 A for twitch c f . A change into the adult pattern was observed in kittens 10-70 weeks of age (Fig. 4 B). 111order to compare twitch parameters in musrles of different age. the relation between the stimulus intemit! and c f or lirt was approximated from the graphs as shown in Fig. 4 and expressed in following may
where r is threshold intensity. c intensity of reach maximal response level x . and 6 the slope of the curve (see Fig. 4 A). A positive slope denotes an increase in cI or hrr with stimulus strength a n d a negative slope the reterse. An analysis of \ariance a n d a calculation of confidence intervals according to the method of Scheff+ (1961) was performed for the 6-values of muscles from cats of different age.
Significant differences were revealed for ct of kittens below ten weeks of age and older cats. However, there were no statistical difference between any age groups with respect to krt changes with stimulus intensity, although the mean values of ct and hrt seem to vary in a similar way with age (Fig. 4 C). The standard deviation for hrt was much larger than for ct. These findings strongly support the idea that the recruitment of motor units to increasing stimulus intensity followed different patterns in young and in adult eye muscles, with a period of transition at 6-10 weeks.
2001
f -
0 1 2
4
6
10
_
c
dlh
Agclmsktl
Fig 5 Changes in fubioii frequency of tetanic contraction with age. Average values plotted; standard dekiation: 2 2 7 5 H7.
TWITCH AND TETANUS I N KITTEN EYE MUSCLE
Fig. 6. Maximal rate of tension rise in tetanic contractions for different age groups. For comparison between age groups, the rate of tension rise for each muscle has been related to the maximal tetanic terision of that muscle (Po)and expressed in percent of P, per msec. Standard deviation & 0.8";.
139
21
Absolute values for ct and hrt at threshold and at maximal activation are given in Table 11. It was found that the threshold C I , but not hrf increased continuously with age. Fusion frequency. As shown in Fig. 5 the fusion frequency was around 200 Hz in the newborn kitten and increased with age until the values saturated at about 325 Hz for muscles older than 6 weeks. The difference between values above 6 weeks of age and those below age 2 weeks was statistically significant (p 20.01). Maximal rate of tension rise. Stimulus frequencies higher than the fusion frequency had to be applied in all age groups in order to attain maximal rate of rise of the tetanic response. In the newborn animal stimulation at 400 Hz was necessary and in the adult cat 600 Hz. Mean values of rate of tension rise in Fig. 6 have been expressed as the tension increase in percent of maximal tetanic tension (Po) per millisecond. There was no significant variation between muscles up to 6 weeks of age. At 10 weeks the values started to increase and at 20 weeks they were even highei than in the adult animal. Effect o f snccinylcholine (Sch). This drug is known to cause a long-lasting contracture in extraocular muscle of the adult cat, probably by inducing an extensive depolarization of the TABLE 111. Effects of succinylcholine on inferior oblique muscles at different ages. Drug action has been expressed as succinylcholine-induced tension in percent of the maximal tetanic tension, and has been measured at the time of maximal succinylcholine effect and 5 and 10 min later. Mean values (& S.D.) are given for each age group. Age 0 week 1 week
2 weeks 4 weeks 6 weeks 10 weeks 20 weeks Adult
Max effect
5 min
10 min
140
GUNNAR LENNERSTRAND AND JERKER HANSON
multiply innervated fibres (Bach-y-Rita 1971). A contracture could be elicited by the drug in kitten inferior oblique muscle of all ages. Drug action was expressed as the Sch-induced tension in percent of the maximuin tetanic tension. There was no significant difference in drug action between age groups (Table 1"). The drug effects 5 and 10 min after the injection were slightly larger in the older animals. Duration of drug action was 10 to 15 min in animals at age 10 weeks and below, and slightly longer (15-20 min) in the older animals.
Discussion Postriutal clifferentiutiati of fast arid slow fibres
At birth the inferior oblique was a much slower muscle than in the adult cat. The speed of twitch contraction increased gradually over at least the first half year of the kitten's life. I n this respect the development of eye muscles was similar to that of fast limb muscles, although in the latter adult values were already obtained at the age of 2 months (Buller et a/. 1960, Buller and Lewis 1965, Mann and Salafsky 1970, Westerman et a/. 1973, Hammarberg and Kellerth 1975 a). However, if speed of contraction in eye muscles instead was estimated from the stimulus frequency to produce a fused response, the mature stage would seem to be reached by 6 weeks. By using the rate of tension rise as the measure of contraction speed, one would conclude that muscle development did not start until about 6 weeks of age and continued to approxiniately age 20 weeks. The differences between the postnatal development of fusion frequency and twitch contraction time might be related to post-tetanic effects. The twitch responses reported in this paper were not influenced by previous muscle activity. It will be shown in a subsequent paper (Lennerstrand and Hanson 1978) that repetitive stimulation prolonged twitch relaxation and induced a staircase phenomenon in the tetanic response. These effects were most marked in the older animals, and could account for most of the levelling off of the curve relating fusion frequency to age. In order to explain the developmental changes in the rate of tension rise of eye muscles one might propose that the slow fibres matured more rapidly than fast fibres. Constant values for the rate of tension rise during the first 6 weeks in spite of a decrease in twitch contraction time would indicate that the slow fibre system completed most of its development during this period. The parallel changes in twitch contraction time and rate of tension rise up to age 20 weeks, suggest that the fast fibres obtain their adult properties much later. Another finding to support the idea that the development of slow and fast fibres of eye muscles followed different time courses was that the time for total twitch relaxation increased from birth to six weeks of age, which would indicate that the slowest fibres, which contribute most to the latest phases of the twitch response, not only remained slow from birth but became still slower with age.
The e j u miscIe resporises to threshold neri>estimulation It was found that increasing the intensity of nerve stimulation from the threshold to the supramaximal level affected twitch responses differently depending on the age of the muscle. In the young muscles, fast fibres seemed to be activated first and as stimulus intensity rose,
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more and more slow fibres were recruited. The same pattern is seen in ordinary skeletal muscle irrespective of age. However, in cat eye muscle of age 10 weeks the response to threshold stimulation was slower than those to supra-threshold stimulus intensities. This pattern is regularly seen in the adult muscle (Lennerstrand and Bach-y-Rita 1974). The muscle fibres responding at threshold must be innervated by large diameter nerve fibres, since these would be activated by the lowest stimulus. It seems unlikely that the muscle fibres in question would be innervated by small nerve fibres in the young animal and later re-innervated by large nerve fibres, or that the nerve fibres to these muscle fibres should have grown at a faster rate than other nerve fibres. Fast hindlimb muscles are known to develop larger nerve fibres than slow muscle (Nystrom 1968 b), as reflected also in the higher conduction velocities of nerve axons innervating fast motor units than slow motor units (Bagust et a1. 1973, Bagust 1974). This relation holds also for kitten hindlimb muscle (Bagust, Lewis and Westerman 1974). Since the number of muscle fibres seems to remain unchanged during postnatal development (Westerman et al. 1973), the most plausible explanation for the appearance in eye muscles of a slow response to threshold nerve stimulation at 10 weeks of age would seem to be that some portion of the muscle fibres had changed their twitch properties and developed slightly slower contractions, in the same manner as the slow fibres in the soleus muscle (cf. Fig. 2 A and B). These fibres may be located in the outer, orbital layer, since findings in the adult cat indicate that the motor response to threshold stimulation arises in orbital fibres (Lennerstrand and Bach-y-Rita 1974, Bach-y-Rita 1975). The histochemical study of postnatal changes in eye muscles (Hanson et al. 1977) has shown that the orbital layer is poorly defined at birth but fully developed at about 10 weeks of age.
Effects of sirccinylcholine The succinylcholine experiments gave similar results at all stages of development. In response to an injection of the same amount of the substance per unit b.wt., the muscles developed contractures that amounted to 40-50% of the maximal tetanic tension. The drug is thought to induce a long-lasting contraction of multiply innervated muscle fibres, i.e. the slow fibre components, by depolarizing the postsynaptic region of the muscle fibre membrane (Bach-yRita 1971). In eye muscle of the adult cat the drug would seem to act on multiply innervated fibres of both the amphibian and the avian types (Bach-y-Rita P t al. 1977). The multiple innervation of adult extraocular muscle is probably only to a very small extent of the polyneuronal type, with overlapping innervation from several nerve fibres to the same muscle fibre (Bach-y-Rita and Lennerstrand 1974). It is well-known, however, that all fibres in kitten hindlimb muscle are polyneuronally innervated at birth, but that this type of innervation has disappeared at 6 weeks of age (Bagust, Lewis and Westerman 1973). The possibility of polyneuronal innervation of eye muscle fibres in young kittens makes it difficult to draw firm conclusions on the development of multiply innervated fibres on the basis of the succinylcholine expts. We wish to thank Stig Danielsson for advice and assistance in the statistical analysis. This study was supported by grants from Karolinska institutets fonder and the Swedish Medical Research Council (grants No. 4751,4719 and 3875).
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