Qualitative and quantitative studies on Japanese Waltzing mice

,I. COMP.

PATH.

1982.

VOL.

92.

533

QUALITATIVE AND ON JAPANESE

N.

T.

M.

JAMES*,

QUANTITATIVE WALTZING

STUDIES MICE

and M. J.

CABRI&~

* Department

of Human Biology and Anatomy, C’ni~‘ersity She@ld SIO ZTN, U.K. 7 Institute of Sports Medicine, Belgrade, Yqoslaka

WILD*

of Shejield,

INTRODUCTION

Japanese Waltzing mice (JWM) suff er from a neurological deficit that results in a characteristic and almost continuous high speed locomotor activity. Due to their high work output, the oxygen uptake of JWM is significantly greater than that of normal mice. Stereological analyses of their lungs have revealed that alveolar and capillary surface areas and the capillary volumes of JWM are significantly greater than those of normal mice (Geelhaar and Weibel, 197 1) whilst the mitochondrial content of their pneumocytes is also known to be increased (Gail, Massaro and Massaro, 1975). Since the increased oxygen uptake and pulmonary adaptation is due to increased muscular activity, attempts were made to quantify some of the properties of the skeletal muscles of JWM. The sizes of their muscle fibres, some properties of the myonuclei and important features of the muscle capillary network w-ere specifically examined. During the preparation of material for the present study some interesting qualitative data were obtained on both extrafusal fibres and the sensory organs of muscles, the muscle spindles. Some preliminary results have previously been reported (James and Wild, 1978; cabrid, James and Wild, 1981). MATERIALS

Animals

AND

METHODS

and Specimen Preparation

Five JWM and 6 control mice of similar age were used in the present study. Each animal was maintained in an individual cage 40 x 18 cm, with a floor of smooth plastic. Theywere allowed food andwater ad libitum.The cageswerekept in a busy animal house at a relatively constant temperature within the range 20 to 22 “C with alternating 12 h periods of artificial light and darkness. Specimens of extensor digitorum longus (EDL) muscleswere selected for study as they are known to contain both type I and type II musclefibres (James, 1980) and are sufficiently small for muscle spindles to be found with relative ease (James and Meek, 1975, 1979). EDL muscles were initially fixed at their resting length with 3 per cent 0.1 M phosphate buffered glutaraldehyde at pH 7.3 so as to enable possiblecomparisons with earlier studies on marine muscle to be carried out. Four small blocks of muscle were removed from the control mid-belly region of each EDL muscle and fixed for a further 4 h in fresh fixative at 4 “C. They were then transferred to 0.1 M phosphate buffer at pH 7.3 to which sucrose had been added to yield a final concentration of 10 per cent. The specimenswere post-fixed in 2 per cent aqueous osmium tetroxide for 1 h at 4 “C, dehydrated in ethyl alcohol and embedded in Araldite. 0021-9975/82/040533+

13 $03.00/O

(0

1982

Academic

Press

Inc.

(London)

Limited

534

N.

T. JAMES~~

al.

Transverse and longitudinally orientated thin and serial semithin (~1 pm) sections were prepared from each muscle specimen and stained by the appropriate techniques for both light and electron microscopy (Pease, 1964). Sampling

and Stereological

Procedures

Sixteen randomly distributed transverse and longitudinal non-overlapping light microscopical fields each ~4.25 x iO-2 mm2 in area were used for each animal and distributed over each of the four blocks per animal. Random sampling fields were selected by setting the horizontal and vertical movement stage micrometers of a Wild M20 microscope with sequential values obtained from a table of random numbers (Lindley and Miller, 1971). Systematic distribution of sampling fields was specifically avoided to avoid possible sampling interactions with anisotropic fibre type distributions within fasciculi. The light microscopical fields were used to make counts of the number of capillaries (PA)I and (PA)!, and nuclei (NA) per unit area of muscle cross-section and also to measure the reconstructed lengths of 100 myonuclei in the serial semi-thin sections. Muscle fibre cross-sectional areas were also measured using light microscopical fields of semi-thin sections. Field images were projected on to the digitizing tablet of a Kontron MOP AM03 Analyser programmed to use simple counting and measuring techniques. Thin sections were examined by electron microscopy for the study of myonuclei and their chromatin patterns at a final print magnification of 4200 and 38 000, respectively. Fifty profiles from both type I and type II fibres were selected randomly for study of chromatin patterns and heterochromatin content. The numerical density of myonuclei, i.e. the number of nuclei per unit volume of muscle (NV), was estimated by the relation iVv=N~/l (Atherton and James, 1980; Cruz-Orive, 1980) where NA is the number of nuclear profiles seen per unit area of muscle cross-section and E the mean length of myonuclei. The mean volume of nuclei, V, was given by the relation fi= Vv/Nv where the volume fraction (Vv) is given by the area fraction (AA), i.e. Vv=&. The volume fraction of heterochromatin within myonuclei and the mean intercept length across heterochromatin and euchromatin as seen in 2-dimensional micrographs were measured using a slight modification of a technique (James, 1979; J ames and Cabric, 1982) used to measure the distribution characteristics of 2-phase mosaics (Pielou, 1964). The method consists of placing a series of transparent screens bearing randomly orientated lines of known length on micrographs and recording whether heterochromatin or euchromatin lies under the end of each test line. Three types of event can occur. Both ends of each test line can lie on either heterochromatin or euchromatin or one end can lie on each type of chromatin. The experimentally measured frequencies of these events for different lengths of test line can be compared with those expected for a random distribution of chromatin by the procedures outlined by Pieiou (1964). The 3-dimensional volume fraction of nuclei occupied by heterochromatin is also given by the procedure as is the mean random intercept length across both heterochromatin and euchromatin. The values give a measure of the 2-dimensional spatial pattern of chromatin. The length of capillaries per unit volume of muscle (Lv) was measured using 2 different quantitative methods. The number of capillaries per unit area of muscle in cross-section, (PA)~, and the number of capillaries per unit area of muscle in longitudinal section, (PA),,, were used to estimate LV by the formulae Lv=(PA)~+(PA)II

(Underwood,

1970)

and Lv=i{ Terms

for the degree

(PA)I+~(PA):I)

of orientation

of the capillaries,

(Weibei,

1980).

Q,, s, (Underwood,

1970) and a

JAPANESE

distribution relation

concentration

WALTZING

parameter,

MICE

535

MUSCLES

K, (Weibel, 1980) were also evaluated

by the

andK=2{1-gi}.

RESULTS

Several interesting qualitative features were noted intrafusal muscle fibres of Japanese Waltzing mice.

in both

extrafusal

and

Extrafusal Cells Type I extrafusal fibres contained numerous large mitochondria seen to be distributed relatively evenly throughout their transverse sections. In addition, extremely large subsarcolemmal accumulations of mitochondria, often occupying large crescentic regions of the fibre [Fig. 1 (a)], were noted in the majority of type I fibres in any muscle transverse section. They seemed to occur independently of the occurrence of adjacent capillaries. Type I and type II fibres seemed to differ more in their cross-sectional areas in JWM than in the controls. Type II extrafusal fibres were often found to contain large characteristic accumulations of tubules [Figs 2(b), (c), (d) and 31. The tubules were never seen in type I fibres nor in any intrafusal muscle fibres. The quantitative results are presented in Tables 1 to 5. Some important TABLE 1 PROPERTIES

: 3 4 5 Mean 5S.E.

OF MYONUCLEI

AND

MUSCLE

FIBRE

Animal number

12.5 9.9

5.59 5.84

2.18 1.45

373 259

820 1197 1100 908.2 110.9

604.5 503.8 428.2 564.2 50.8

11.8 IO.4 10.2 10.9 0.5

5.12 4.84 4.20 5.12 0.23

1.45 1.48 1.09 1.52 0.18

283 299 259 294 21

1 2 3 4 5 Mean + S.E.

AND

(PA)1

.~

MICE

554.2 730.5

controls (P < 0.0 1). controls (P < 0.05). in the text. TABLE

(L,)

WALTZING

569 855

* Significantly different from t Significantly different from Column headings are defined

LENGTHS

SIZES IN JAPANESE

mme2 .~~~ 856 982 1209 705 680 886 97

RELATIVE

ORIENTATION

(Pn)ll

L”*

mme2

mm mmm3

579 403 406 428 352 433 38

1436 1385 1612 1134 1033 1320 105

Column headings and methods * Underwood (1970), t Weibel

2

OF CAPlLLARIES

of calculation (1980).

are given

(R,,

3, K)

-hi

mm mm-3 1343 1192 1347 1041 923 1168 84 in the text.

IN JAPANESE

n 1, 3,*

WALTllNG

MICE

xt

(0) 0.19 0.42 0.49 0.24 0.32 0.34 0.06

0.65 1.18 1.33 0.79 0.96 1.02 0.12

536

?g.

N.

1. (a) Typical chondria mitochondria. chondria.

extrafusal present x 3900. x 8900. are

type I muscle throughout (b) Intrafusal

T.

JAMES

et at.

fibre of Japanese Waltzing Mice the fibre. Note the subsarcolemmal muscle fibre showing characteristic

(JWM).

Many mitoaccumulation of “clumping” of mito-

JAPANESE

WALTZ1

NG ,MICE -.

MUSCLES

Fig. 2. (a) Transverse section through polar region of muscle spindle of JWM. Two fibres possessing a higher mitochondrial content than other filbres are seen to contain distended lateral sacs of triads. x3500. (b), (c) and (d) Examples o f accumulations of tubules in type II muscle fibrrs. x 43 500, x 7800 and 10 800, respectively.

538

N.

T.

JAMES

et

al.

I Gg. 3. (a) Typical though small accumulation of tubules. Note the presence of few mitochondria in the muscle fibre of JWM clearly demonstrating that it is a type II muscle fibre. x34 000. (b) Typical larger accumulation of tubules. Note that in some regions the tubules are cut transversely but in others they are cut obliquely; consequently they cannot be regarded as vesicles. X 20 000.

JAPANESE

WALTZING

MICE

539

MUSCLES

results can be summarized as follows. The mean cross-sectional area of muscle fibres in JWM is larger than in control muscles (Tables 1 and 4, respectively). The capillary densities (Lv) (Table 2) and myonuclear frequencies (N;) are also greater in JWM than in controls (Table 5). The nuclei of type I and type II fibres in JWM seem to be very similar, as indicated by their heterochromatin content and measurements of the mean intercept lengths across heterochromatin and euchromatin (Table 3). TABLE QUANTITATIVE

ANALYSIS

OF NUCLEAR

Type

3

HETEROCHROMATIN

IN JAPANESE

WALTZING

MICE

Type I I nucleus

I nucleus

Hc-Mean intercept

EL-Mean intercept

H,-Mean intercept

EL-Mean intercept

Animal number

per cent

Pm

pm

per cent

pm

pm

1 3 4 5 M.?an &S.E.

35.6 37.0 34.3 26.8 30.7 33.4 1.8

0.64 1.12 0.55 1.05 0.97 0.77 0.11

0.73 1.37 1.92 2.16 1.86 1.63 0.25

35.4 34.8 32.0 38.7 32.7 34.4 1.2

0.99 0.89 1.45 0.36 0.48 0.78 0.19

1.26 1.19 0.86 1.66 2.17 1.61 0.22

Vv-Hc

Column

headings

are defined

Vv-Hc

in the text.

TABLE PROPERTIES

Nude Animal number

-

OF MYONUCLEI

AND

4

MUSCLE

FIBRE

SIZES IN CONTROL

MICE

jbre

area -..__.-__

pm2

;

403.0 82 1.2

529 479

12.4 12.9

4.11 3.85

0.72 1.09

283 17.5

3 4

541.6 717.9 683.1 728.4 946.2 61.6

403 416 447 508 464 21

10.9 11.4 11.1 12.2 11.8 0.3

3.68 3.65 4.03 4.16 3.91 0.09

0.72 1.05 0.97 1.06 1 .oo 0.07

195 287 241 255 239 19

5

6 Mean IfS.E.

* Significantly different from JWM (P
TABLE LENGTH

Animal number

: 3 4 5 6 Mean &S.E.

(L,)

(PA)

AND

RELATIVE

ORIENTATION

(PA)

JL*

mme2

mm-a

mm mmw3

655 579

365 390 378 466 323 409 389 20

1045 945 1259 1222 947 1150 1095 56

882 757 624 741 706 45

Column headings and methods * Underwood ( 1970)) t Weibel

of calculation ( 1980).

5

OF CAPILLARIES

are given

(a,,

S K)

IN CONTROL

MICE

Kt

-Lt

mm mm-3 873 957 1092 1126 847 1039 989 47 in text.

0.23 0.25 0.40 0.24 0.32 0.29 0.29 0.03

0.74 0.81 1.14 0.77 0.96 0.90 0.90 0.06

540

N.

T.

JAMES

et

al.

Gg. 4. (a) Transverse section of equatorial region of muscle spindle of JWM containing 2 large nuclear hag fibres and 2 smaller nuclear chain fibres. Note the presence of a capillary (containing an electron-dense red blood cell) lying within the periaxial space. x 1450. (b) Transverse section of the capillary seen above. Note microvilli on the endothelial cell surface facing the capillary I~mwn and also the electron-dense body within the endothelial cytoplasm. x 13 500.

JAPANESE

WALTZING.

MICE

MUSCLES

541

In trafusal Cells Prominent clumping of mitochondria was usually present in the two smaller intrafusal fibres, presumably nuclear chain fibres, which were present in each fibres were also found to contain muscle spindle [Fig. 1(b)]. Th ese intrafusal numerous triads in which the lateral sacs seemed considerably more distendecl than in normal mouse intrafusal fibres [Fig. 2(a)]. Twelve muscle spindles were examined in the EDL muscles of JWM. Four spindles contained a capillary within the periaxial space [Fig. 4(a)]. Each capillary possessed a reduplicated basement membrane and was always surrounded by a complete investing layer of perineural epithelial cells [Fig. 4(b)]. The endothelial cell surfaces facing the capillary lumen seemed to bear numerous microvilli which could be identified in both longitudinal and transverse section. Intracytoplasmic electron-dense structures were also noted [Fig. 4(b)].

Kg.

5. Transverse section of equatorial bag fibres and 2 smaller nuclear axial space. X 950.

region of muscle spindle of JM:M containing chain fibres. Note the absence of any capillary

2 large nuclear from the prri-

DISCUSSION

The results tJWM contain

clearly indicate that the muscle fibres of the EDL muscle of some unique structures and also that some of the intracellular

542

N.

T.

JAMES&

cd.

components differ quantitatively from those of normal mice. The currently used JWM belong to a large group of Waltzershaker mouse mutants the prototype of which reached European and American laboratories from the Far East toward the end of the nineteenth century. After much study (Grtineberg, 1952) the most important mutant is known to be the Waltzer (v). Its behavioural abnormalities consist of rapid circular movements, maintained for long periods, head shaking in the sagittal plane, deafness and usually an inability to swim. These can usually be recognized by the end of the second week of life and the Va/Va homozygotes are more severely affected than the Va/+ heterozygotes (Grtineberg, 1956). The cochlea characteristically undergoes early postnatal degeneration and its pathology, first studied by Lennep (1910, lot. cit. Grtineberg, 1956) has been summarized by Gruneberg (1956). The mutant is useful in that it provides a ready source of almost continuously exercised muscle which is not thought to be associated with any intrinsic pathology. The occurrence of numerous mitochondria in muscle fibres, particularly in those of type I, is entirely consistent with the increase in mitochondrial content previously seen in exercised muscles (Holloszy, 1967 ; Gollnick and King, 1969 ; Kowalski, Gordon, Martinez and Adamek, 1969; Kraus, Kirsten and Wolff, 1969 ; Gollnick, Tonuzzo and King, 197 1; Kiessling, Piehl and Lundquist, 197 1; Morgan, Cobb, Short, Ross and Gunn, 1971; Hoppeler, Ltithi, Claassen, Weibel and Howald, 1973; Prince, Hikada, Hagerman, Staron and Allen, 198 1; James, 198 1). Similarly, the apparently higher mitochondrial content of intrafusal fibres within muscle spindles reflects an increased work output (Mahon and James, 1975). Of particular interest are the accumulations of tubules seen in type II muscle fibres. Their significance is unclear at present. Numerous reports of accumulations of tubules in clearly diseased muscles have been published (Mair and Tome, 1972) though none has been described as either being similar in structure or possessing a similar distribution. One study has reported an association of tubules with human type II fibres but these occurred almost exclusively in cases of hypo- and hyperkalaemic paralysis (Engel, Bishop and Cunningham, 1970). The present authors, however, have seen similar accumulations of tubules in skeletal muscles which have been forced to undergo compensatory hypertrophy consequent upon the surgical removal of synergistic muscles (manuscript in preparation). It is worth noting that the extremely rapidly contracting muscles of the bat larynx which contract for long periods contain extensive tubular bundles (Cho, Sidie and De Bruyn, 1972). Currently, however, it is not clear whether the tubules possess pathological significance. The muscle spindles of JWM are clearly different from those of normal mice. First, the capillaries found in the Two findings merit detailed consideration. periaxial space have not previously been described as a frequent occurrence in laboratory animals smaller than the rabbit (Banks and James, 1973a; James and Meek, 1979). It is possible that the presence of periaxial capillaries is related to the increased oxygen demand of intrafusal fibres due to greater spindle activity and increased mitochondrial content. The capillary endothelial cells are of an unusual type for skeletal muscles in that numerous micro-villi are present on the surface facing the capillary lumen rather than a

JAPANESE

WALTZING

MICE

MUSCLES

543

relatively smooth surface typical of the capillaries that occur in either spindle capsules or between extrafusal fibres. Such capillary microvillous surfaces have been recorded for a variety of tissues (Cliff, 1976) and their significance is obscure. Second, the distended lateral sacs of the intrafusal triads and pentads seem larger and more numerous than in normal mice though quantitation was not attempted. The large distended lateral sacs of JWM are more typical of intrafusal fibres in animals larger than the mouse (James and Meek, 1973 ; Banks and James, 1973b), though smaller sacs occur frequently in both normal and dystrophic muscle (James and Meek, 1975, 1979). As it has previously been suggested that the distended sacs are responsible for the synthesis and secretion of the mucopolysaccharide which is known to occur in the periaxial space (Brzezinski, 1961), their occurrence should not be regarded as pathological. Much of the quantitative data obtained in the present study indicate that muscles of JWM appear to possess properties very similar to those of exercised or hypertrophic muscles. For example, the increase in capillary length (Lv) is entirely consistent with increases previously reported (James, 198 1). The differences between the nuclei of type I and type II fibres which are normally present in muscles of mixed fibre composition are not present in JWM muscle. Similar findings have been reported in exercised dog and mouse muscles (Cabric’ and James, 1982) as have increases in the nuclear density (NV). In addition the relatively low volume fraction of heterochromatin is also characteristic of exercised muscle. It is suggested that the use of JWM could be of great value in studying the reaction of skeletal muscle to prolonged exercise. SUMMARY

The extensor digitorum longus muscles of Japanese Waltzing mice (JWM) were subjected to a combined quantitative and electron microscopical analysis. Numerous changes were found which indicated hypertrophic changes of exercised muscles. Some of the muscle spindles found were atypical in that some were vascularized and many more larger sacs of the triads and pentads were observed. A characteristic finding exclusive to type II muscle fibres was the frequent occurrence of large numbers of small tubules forming distinct and discrete accumulations. The quantitative cytological data suggest that the muscles of JWM show typical features of muscular adaptation to exercise: and that these mutants are convenient for the study of chronic muscular hypertrophy. ACKNOWLEDGMENT

The authors wish to thank P.M.S. (Instruments) Ltd for the loan of a 16K Pet Gommodore microprocessor and digitizing tablet necessary for the quantification of nuclear chromatin contents. REFERENCES

Atherton, G. W., and James, N. T. (1980). Stereological analysis of the number nuclei in skeletal muscle fibres. Acta Anatomica, 107, 236-240.

of

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et d.

Banks, R. W., and James, N. T. (1973a). The blood supply of rabbit muscle spindles. Journal of Anatomy, 114, 7-12. Banks, R. W., and James, N. T. (1973b). The fine structure of the guinea-pig muscle spindle. Zeitschrift ftir Zellforschung und Mikroskopische Anatomie, 140, 357-368. Brzezinski, K. D. (1961). Untersuchungen zur Histochemie der Muskelspindeln. II. Zur topochemie und Funktion des Spindel und der Spindelkapsel. Acta histochemica, 12, 277-288. Cabrid, M., and James, N. T. (1982). Morphometric analysis of the muscles of exercise trained and untrained dogs. American Journal of Anatomy (in press). cabrid, M., James, N. T., and Wild, M. J. (1981). Quantitative studies on Japanese Waltzing mice. Journal of Anatomy 133, 695. Cho, Y., Sidie, J. M., and De Bruyn, P. P. M. (1972). Electron microscopical studies on a tubular filamentous fasciculus in the bat cricothyroid muscle, Journat of Ultrastructure Research, 41, 344-357. Cliff, W. J. (1976). Blood Vessels. Cambridge University Press, Cambridge. Cruz-Orive, L.-M. (1980). On the estimation of particle number. Journal of Microscopy, 120, 156-167. Engel, W. K., Bishop, D. W., and Cunningham, G. G. (1970). Tubular aggregates in type II muscle fibres: ultrastructural and histochemical correlation. Journal of Ultrastructure Research, 31, 507-525. Gail, D. B., Massaro, G. D., and Massaro, D. (1975). Intraspecies differences in lung metabolism and granular pneumocyte mitochondria. Respiratory Physiology, 23, 175-180. Geelhaar, A., and Weibel, E. R. (1971). Morphometric estimation of pulmonary diffusion capacity. III. The effect of increased oxygen consumption in Japanese Waltzing mice. Respiratory Physiology, 11, 354-366. Gollnick, P. D., and King, D. W. (1969). Effects of exercise and training on mitochondria of rat skeletal muscle. American Journal of Physiology, 216, 1502-1509. Gollnick, P. D., Tonuzzo, C. D., and King, D. W. (1971). Ultrastructural and enzyme changes in muscles with exercise. In Muscle Metabolism During Exercise. B. Pernow and B. Saltin, Eds, Plenum Press, New York, pp. 69-85. Griineberg, H. (1952). The Genetics of the Mouse, 2nd edition. Nijhoff, The Hague. Grtineberg, H. (1956). Hereditary lesions of the labyrinth in the mouse. British Medical Bulletin, 12, 153-157. Holloszy, J. 0. (1967). Biochemical adaptations in muscle. Effects of exercise on mitochondrial uptake and respiratory enzyme activity in skeletal muscle. Journal of Biological Chemistry, 242, 2278-2283. Hoppeler, H., Luthi, P., Claassen, H., Weibel, E. R., and Howald, H. (1973). The ultrastructure of the normal human skeletal muscle. A morphometric analysis on untrained men, women and well-trained orienteers. Pj%gers Archiv, 344,2 17232. James, N. T. (1979). Studies on the fibre type patterns in skeletal muscles. II. A spatial autocorrelation analysis. Journal of Anatomy, 129, 221-222. James, N. T. (1980). Quantitative studies on the contiguity of muscle fibre types in normal and hypertrophic skeletal muscles. Acta Anatomica, 108, 132-136. James, N. T. (1981). A stereological analysis of capillaries in normal and hypertrophic muscle. Journal of Morphology, 168, 43-49. analyses of normal and hyperJames, N. T., and Cabric, M. (1982). Q uantitative trophic extensor digitorum longus muscles in mice. Experimental Neurology, 76,. 284-297. James, N. T., and Meek, G. A. (1973). Studies on the sarcoplasmic reticulum of rat, cat and sheep intrafusal fibres. Journal of Anatomy, 116, 219-226. James, N. T., and Meek, G. A. (1975). Ultrastructure of muscle spindles in dystrophic mice. Nature, 254, 612-613. James, N. T., and Meek, G. A. (1979). Ultrastructure of muscle spindles in C57BL/ 6Jdy2J/dy2J dystrophic mice. Exjerientia, 35, 108-109.

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MUSCLES

54.5

James, N. T., and Wild, M. J. (1978). A study of the capillaries of Japanese Waltzing mice. Journal of Anatomy, 127, 220. Kiessling, K. H., Piehl, K., and Lundquist, C.-G. (1971). Effect of physical training on ultrastructural features in human skeletal muscle. In Muscle Metabolism During Exercise. B. Pernow and B. Saltin, Eds, Plenum Press, New York, pp. 97 101. Kowalski, K., Gordon, E. E., Martinez, A., and Ademek, J, (1969). Changes in enzyme activities of various muscle fibre types in rat induced by different exercises. Journal of Histochemistry and Cytochemistry, 17, 601-607. Kraus, H., Kirsten, R., and Wolff, J. R. (1969). Die Wirkung von Schwimmund Lauftraining auf die cellulare Funktion und Struktur des Muskels. l’jGger.r L4rchiv, 308, 57-65. Lindley, D. V., and Miller, J. C. P. (1971). Cambridge Elementary Statistical TableJ. Cambridge University Press, Cambridge. Mahon, M., and James, N. T. (1975). Studies on muscle spindles in normal and hypertrophic muscles. Journal of Anatomy, 120, 611-612. Mair, W. G. P., and Tome, M. S. (1972). Atlas of the 1Jltrastructure of Diseased Human *Cluscle. Churchill-Livingstone, Edinburgh. Morgan, T. E., Cobb, L. A., Short, F. A., Ross, R., and Gunn, M. (1971). Effects of long-term exercise on human muscle mitochondria. In Muscle Metabolism During Exercise. B. Pernow and B. Saltin, Eds, Plenum Press, New York, pp. 87-95. Pease, D. C. (1964). Histological Techniques for Electron Microscopy. Academic Press, London. Pielou, E. C. (1964). The spatial pattern of two phase patchworks of vegetation. Biometrics, 20, 156167. Prince, F. P., Hikada, R. S., Hagerman, F. S., Staron, R. S., and Allen, W. H. (1981). A morphometric analysis of human muscle fibres with relation to fibre types and adaptations to exercise. Journal of the Neurological Sciences, 49, 165-179. Underwood, E. E. (1970). Quantitative Stereology. Addison-Wesley, Massachusetts. Weibel, E. R. (1980). Stereological Methods, Volume 2, Theoretical Foundations. Academic Press, London. [Received for publication,

Ju& 3 lst, 198 l]