J. Anat. (1989), 163, pp. 191-200 191With 7 figures

Printed in Great Britain

Intrafusal muscle fibre types in frog spindles*

F. H. DIWANt AND F. ITODepartment of Physiology, Nagoya University School of Medicine,

Nagoya 466, Japan

(Accepted 19 July 1988)

INTRODUCTION

It is now firmly established that the intrafusal muscle fibres of mammalian musclespindles are classified into three types: nuclear bag,, nuclear bag2 and nuclear chainfibres (Barker & Banks, 1986). This classification is based on histochemical,morphological, ultrastructural and physiological studies (Ovalle & Smith, 1972;Barker et al. 1976; Banks, Harker & Stacey, 1977; Saito, Tomonaga & Narabayashi,1977; Boyd, 1980).Amphibian muscle spindles have not received such intensive study as those of

mammals. In frog spindles at least two types of intrafusal fibre, a large twitch fibre anda small, non-twitch fibre are thought to be present (Barker, 1974; Ovalle & Smith,1975). Recently, Yoshimura et al. (1987) examined the histochemical properties of thefrog intrafusal muscle fibres and suggested that they have a similar pattern to those ofmammalian spindles. In this study, some morphological and ultrastructuralcharacteristics of the frog intrafusal muscle fibres are investigated. A preliminaryaccount of this study has already been reported (Diwan, Yoshimura & Ito, 1987).

MATERIALS AND METHODS

The dorsal and ventral semitendinosus (dST and vST), iliofibularis (IF) andsartorius (SR) muscles of the bullfrog Rana catesbeiana were used. The frogs werepithed after being anaesthetised by an injection (1 ml/100 g) of 10% ethyl carbonateinto the dorsal lymphatic sac. The muscles were kept at their in situ lengths duringfixation. For light microscopy, 24 muscles (6 of each type) were prepared according tothe silver impregnation technique of Barker & Ip (1963), modified by Boddy & Diwan(unpublished) as follows: (i) After fixation, the muscles were washed at a flow rate of1 litre/h for 24 hours in 0-02% aluminium sulphate made up to pH 9 with a saturatedsolution ofNaOH. (ii) After 30 hours in ammoniacal alcohol the muscles were dippedrepeatedly into a 1 % agar solution. The agar coating was then left to gel. (iii) Themuscles were incubated by suspending them for 7 days in freshly prepared 1 5% silvernitrate in the dark at 37 °C in a shaking water bath. The agar coating was thenremoved before placing the muscles in the reducer for 24 hours.

Muscle spindles were teased from silver stained muscles and examined with the lightmicroscope. Measurements were made with an eyepiece scale at a final magnificationof x 200 and were standardised to the nearest micrometer.For electron microscopy, 8 muscles (2 of each type) were fixed, at in situ length, in

a solution of 0-2 M cacodylate buffer, pH 7-4. After 30 minutes the muscles were* Reprint requests to F. Ito.t Present address: Department of Biology, College of Science, University of Basrah, Iraq.

192 F. H. DIWAN AND F. ITO

A

B

D

E

F _ __ __ __ ____ __ __ __

F

H =

Fig. 1. Schematic diagrams representing examples of simple type spindles teased from silver-stainedfrog hindlimb muscles. Intrafusal muscle fibres are shown as horizontal lines and capsules as fusiformswellings. The vertical bars represent the musculotendinous junctions in the region of the spindles.A, single spindle; B, parallel spindles; C-F, tandem spindles; C, with two separated capsules; D, withtwo joined capsules; E, with an intrafusal bundle excluded from one capsule; F, with an intrafusalfibre excluded from one capsule; G and H, spindle systems; G consists of one single along withtandem spindles; H consists of two tandem spindles.

transversely cut and then longitudinally sliced and fixed for a further 90 minutes infresh fixative, followed by 2 hours postfixation in 1% osmium tetroxide in cacodylatebuffer, before dehydration and embedding in Araldite. Muscle spindles were locatedin cross-sections stained with methylene blue. Eighteen muscle spindles were seriallyand transversely or longitudinally cut in alternate thin and ultrathin sections. Theultrathin sections were stained with uranyl acetate and lead citrate and examined witha Hitachi 500 electron microscope.

RESULTS

Spindle typesA noticeable feature of the frog muscle spindle is that it extends throughout the

length of the muscle. By using the silver impregnation technique it was possible toclassify the spindles into two main types, namely 'simple' and 'complex' spindles.Simple spindles were those that consisted of one or more bundles of intrafusal musclefibres regardless of the number of capsules they may have as long as these enclose all

Frog spindle

A

B

C

D

E

FI

Fig. 2. Schematic diagrams representing examples ofcomplex type spindles teased from silver-stainedfrog dorsal semitendinosus and iliofibularis muscles. Intrafusal muscle fibres are shown as horizontallines and capsules as fusiform swellings. The vertical bars represent the musculotendinous junctionsin the region of the spindles. The intrafusal bundles are enclosed by at least one common capsule (seetext). Note the short intrafusal fibres in A, C, E, and F.

or some of the bundles and provided that bundles or individual intrafusal fibres,excluded from some capsules, did not have their own separated capsules. In this way,single, parallel and tandem spindles were regarded as simple types. Tandem spindleswith up to four capsules were seen and some had two joined capsules. Few spindlesystems (Gray, 1957) were found and these were regarded as a simple type becausethey did not have a common capsule. As described by Barker (1974), they consistedof tandem spindles, and sometimes 1-2 single spindles, lying together in parallel toform a bundle of spindle units.Complex spindles were those that consisted of two or more intrafusal bundles

enclosed by at least one common capsule. In addition, individual bundles had theirown capsule or a shared capsule with only some of the other bundles. Therefore,complex spindles were of different combinations of intrafusal bundles. Examples ofsimple and complex spindles are shown schematically in Figures 1 and 2, respectively.Simple spindles were found in all muscle types whereas complex spindles were confinedto the dST and IF muscles.

Number of spindles and intrafusal muscle fibresThe number of spindles varied among individual muscles of each type. As shown in

Table 1, the mean numbers of spindles in the dST (20 66), vST (18-83) and IF (24 66)

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F. H. DIWAN AND F. ITO

Table 1. Numbers of spindles with various combinations of intrafusal fibre types (L,large; M, medium; S, small) in four frog hindlimb muscles (spindle numbers are totalof six muscles)

dST vST IF SR

LMS 98 (79 03 %) 84 (74.33 %) 103 (69 59%) 40 (68&96%)LM 10 (8-06%) 8 (7 07%) 23 (15-54%) 11 (18-96%)LS 16(12-90%) 21(18-58%) 18(12-16%) 7 (12-06%)L 4 (2-70%)Total 124 113 148 58Spindles/muscle 20-66 18-83 24-66 9-66

Table 2. Mean polar diameters of intrafusal fibre types in four frog muscles.All measurements in ,um. In parentheses are the numbers of intrafusal fibres in spindles sampled

from six muscles.

dST vST IF SR

Large 25 09 (148) 25-39 (128) 26-43 (159) 29-37 (59)Medium 17 27 (163) 15-77 (116) 16-6 (175) 18-16 (68)Small 9-48 (308) 8-66 (300) 9 12 (283) 8 95 (63)Total number 619 544 617 190Fibres/spindle 4 99 4-81 4-16 3-27

muscles were higher than the mean number in SR muscles (9 66). The number ofintrafusal muscle fibres also varied considerably among individual spindles in allmuscle types. Four spindles teased from IF muscles were found to contain a singleintrafusal fibre only. In contrast, the highest number (18) of intrafusal fibres was foundin a spindle from a dST muscle. The mean numbers of intrafusal fibres in the spindlesof dST, vST and IF were 4 99, 4-81 and 4-16, respectively and it was smaller (3 27) inSR spindles (Table 2). The intrafusal muscle fibres were lying as one bundle in SRspindles, whereas in those of other muscles they were arranged in 1-4 bundles, eachconsisting of 2-5 intrafusal fibres.

Diameter of intrafusal muscle fibresAs mentioned before, frog intrafusal muscle fibres are usually as long as their

extrafusal counterparts. Short intrafusal fibres were occasionally found, in somespindles only. The capsule (in single spindles) was usually located on either side of themidpoint between the origin and insertion of the intrafusal fibres. In some spindles,intrafusal fibre diameters varied between the two poles with respect to the location ofthe capsule. The diameter was generally smaller at the short pole than at the long poleand the difference was more evident in spindles with a capsule located near theirextreme ends. In such cases, all the intrafusal fibres had a comparable and thindiameter at the short pole and a much thicker diameter at the long pole.

In general, the diameter of individual intrafusal fibres was relatively constant for along distance in the polar region next to the spindle capsule. Measurements wereusually taken in this region. The intrafusal muscle fibres were classified into three typeswith a large, medium and small diameter. Figures 3-6 show diameter histograms of theintrafusal fibres in the four muscles. Although overlaps between the diameter rangesare shown in the histograms, it should be noted that such an overlap arose onlybetween different spindles and the three types were almost always distinguishable in

194

Frog spindle

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Diameter (Lim)Fig. 3. Polar diameter histograms of frog intrafusal muscle fibres; a sample of 619 intrafusal fibresbelonging to 124 spindles from 6 dorsal semitendinosus muscles. The left histogram is broken downon the right in terms of the 308 small, 163 medium, and 148 large fibres in the sample. Measurementsmade on teased silver-stained spindles.

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Fig. 4. Polar diameter histograms of frog intrafusal muscle fibres; a sample of 544 intrafusal fibresbelonging to 113 spindles from 6 ventral semitendinosus muscles. The left histogram is broken downon the right in terms of the 300 small, 116 medium, and 128 large fibres in the sample. Measurementsmade on teased silver-stained spindles.

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196 F. H. DIWAN AND F. ITO

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Diameter (gm)Fig. 5. Polar diameter histograms of frog intrafusal muscle fibres; a sample of 617 intrafusal fibresbelonging to 148 spindles from 6 iliofibularis muscles. The left histogram is broken down on the rightin terms of the 283 small, 175 medium, and 159 large fibres in the sample. Measurements made onteased silver-stained spindles.

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Fig. 6. Polar diameter histograms of frog intrafusal muscle fibres; a sample of 190 intrafusal fibresbelonging to 58 spindles from 6 sartorius muscles. The left histogram is broken down on the rightin terms of the 63 small, 68 medium, and 59 large fibres in the sample. Measurements made on teasedsilver-stained spindles.

any individual spindle. Some of the extreme values shown in the histograms werecaused, as mentioned above, by the site of capsule location in some spindles. The meandiameters of the large intrafusal fibres in the dST (25 09 ,um), vST (25-39 ,um) and IF(26A43 /tm) spindles were comparable, while it was higher (29-37 ,um) in SR spindles(Table 2). The mean diameters of the medium intrafusal fibres were comparable(1 5-77-18 16 ,um) in the spindles from all muscles. The diameters of the small intrafusalfibres were also comparable (8&66-9 48 ,um).

Fig. 7 (A-B). Electron micrographs of a transverse section through the reticular zone of a dorsalsemitendinosus muscle spindle showing types of intrafusal fibres. This spindle has two intrafusalbundles; (A) consists of a large bag fibre (L), medium bag fibre (M) and a compact chain fibre (cC);(B) consists of a large bag fibre (L) and a reticular chain fibre (rC).

Frog spindle 197

F. H. DIWAN AND F. ITO

As the intrafusal fibres are arranged in bundles, it is convenient to describe thecombinations of intrafusal fibre types in terms of bundles. Spindles with a highnumber of intrafusal fibres consisted of up to 4 bundles. Various combinations ofintrafusal fibre types were observed in intrafusal bundles, the most frequentlyencountered comprising one large fibre, one medium and one to two small fibres.Spindles with only one large intrafusal fibre were rare, while those lacking eithermedium or small intrafusal fibres were common (Table 1).

Electron microscopic observationsSerial transverse sections of spindles from all studied muscle types revealed that the

intrafusal fibres were not only different in their diameters but also in terms of centralnucleation and the form of the reticular zone (Fig. 7). Accordingly, four types ofintrafusal muscle fibre were identified. (i) A large diameter intrafusal fibre with areticular zone containing a relatively large bag of up to 4 nuclei abreast. (ii) A mediumdiameter intrafusal fibre with a reticular zone containing a small bag of up to 3 nucleiabreast. (iii) A reticular chain fibre, which has a small diameter and a reticular zonecontaining a nuclear chain. (iv) A compact chain fibre, which has a small diameter butlacks a reticular zone and has a chain of nuclei embedded in a typical compactzone.

In the sensory regions, there was a general decrease in the diameter of all intrafusalmuscle fibres due to a reduction in their myofibrillar contents. However, differences indiameter could still be recognised among intrafusal fibre types. The short intrafusalfibres, mentioned earlier, were of the small type and some had a reticular zone. Theyusually lay within the capsule limits or ended a short distance from one or both sidesof the capsule.The nuclei in the reticular zone, particularly those of the large and medium

intrafusal fibres, were long and exhibited many lobes and nuclear processes. Satellitecells were numerous in the sensory compact zone, while a few were found in thereticular zone. In cross-sections, 3-5 satellite cells were usually present aroundindividual intrafusal fibres, marking the transition region between the compact andreticular zones.

In the sensory region, intrafusal bundles were enclosed by separate inner capsulesheaths. Each bundle had its own compartment in the outer capsule or shared one withanother bundle. In this way, the periaxial space of the common capsule could bedivided into up to four subcompartments. It is worth noting that a large intrafusalfibre was always present in each intrafusal bundle and, in tandem and complexspindles, it usually had a small nuclear bag or a nuclear chain in capsules other thanthe common capsule in which it had the large nuclear bag.

DISCUSSION

Early studies on frog muscle spindles (Gray, 1957; Barker & Cope, 1962; Smith,1963; Page, 1966; Brown, 1971; Ovalle & Smith, 1975; Sterling, 1975) have generallyagreed that the intrafusal muscle fibres are of two types with large and small diameter.However, these studies were carried out before the mammalian intrafusal complementwas clarified.The present study showed that almost all the intrafusal muscle fibres extend

throughout the muscle length and short intrafusal fibres were only occasionally found.It was possible, despite regional variations, to recognise three distinct classes of large,medium and small itnrafusal fibre diameter. In the equator, these fibre types were also

198

Frog spindle 199different in their central nucleation arrangement. In the reticular zone, the large andmedium intrafusal fibres have large and small nuclear bags, respectively. Although allthe small intrafusal fibres have a chain of nuclei, they can be subdivided into reticularchain and compact chain fibres according to whether they possess a reticular zone ornot.

Barker & Cope (1962) have described the increase in the number of central nucleito "occur at most no more than three abreast in the large fibres, but do not aggregateinto definite nuclear bags". Besides, the small fibres have fewer nuclei and resemble themammalian nuclear chain fibres, though the arrangement is less regular. Barker &Cope (1962) used the light microscope to examine paraffin-embedded and seriallysectioned fourth toe muscles of a much smaller frog species Rana rugulosa and Ranaguentheri weighing only 120-200 g as compared with the 500 g weight of Ranacatesbeiana frogs used in this study. Although it was not possible to decide the centralnucleation arrangement in our silver-stained spindles, nuclear bags and chains wereeasily recognisable in semithin sections stained with methylene blue.An electron micrograph published by Karlsson and his colleagues (Fig. 2 in

Karlsson, Hooker & Bendeich, 1971; Fig. 8 in Bendeich, Hooker & Karlsson, 1978)shows an intrafusal muscle fibre that has a nuclear bag. However, they attributed theaggregation of nuclei to the slackened condition of the muscle and maintained that instretch the nuclei rearrange themselves into a single row. If this holds true for thisstudy, although the muscles and species used were also different it is difficult tounderstand why the aggregation is confined to the large and medium diameterintrafusal fibres only. The present study demonstrated the nuclear bag even when themuscles were kept at their in situ length during fixation.

According to Page (1966), the majority of the small intrafusal fibres do not possessa reticular zone and such fibres were also observed by Katz (1961). On the other hand,Karlsson (1972) had never observed a muscle fibre without a reticular zone and he wasnot in favour of the differentiation of intrafusal fibres into several types. The presentstudy shows that some of the small intrafusal fibres completely lack a reticular zonewhereas other fibres lack it only in one sensory region but not in another.

Unlike the spherical, oval or lenticular shapes of nuclei in mammalian intrafusalfibres (cf. Barker, 1974), the nuclei in the reticular zone of frog intrafusal fibres appearirregular and have many lobes of varied sizes and shapes. This may reflect the reticularzone organisation and it may be possible that the shape of the nuclei is affected, tosome extent, by the stretch and contraction of the intrafusal fibres.

In conclusion, it is suggested that frog intrafusal fibres are of four types; large andmedium nuclear bag fibres (possibly corresponding to mammalian nuclear bag2 andbag, fibres, respectively) and two types of small nuclear chain fibres, one with and theother without a reticular zone. Obviously, a more detailed and combinedultrastructural and histochemical study is needed.

SUMMARY

Muscle spindles from bullfrog semitendinosus, iliofibularis and sartorius muscleswere examined with light and electron microscopy. Four types of intrafusal musclefibre were identified according to their diameter, central nucleation and reticular zonearrangement: a large nuclear bag fibre, a medium nuclear bag fibre, and two types ofsmall nuclear chain fibres with and without a reticular zone, respectively. It issuggested that they are comparable to the nuclear bag,, bag2 and chain fibres inmammalian muscle spindles.

200 F. H. DIWAN AND F. ITO

This work was supported in part by the Grant-in-Aid for General ScientificResearch from the Ministry of Education, Science and Culture of Japan (61480106).

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BARKER, D. (1974). The morphology ofmuscle receptors. In Handbook ofSensory Physiology (ed. C. C. Hunt),vol. III/2, pp. 1-190. Berlin: Springer Verlag.

BARKER, D. & BANKS, R. W. (1986). The muscle spindle. In Myology (ed. A. G. Engel & B. Q. Banker), pp.309-341. New York: McGraw-Hill.

BARKER, D., BANKS, R. W., HARKER, D. W., MILBURN, A. & STACEY M. J. (1976). Studies of thehistochemistry, ultrastructure, motor innervation and reinnervation of mammalian intrafusal muscle fibers.Progress in Brain Research 44, 67-88.

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KARLSSON, K. (1972). The frog muscle spindle: Ultrastructure and intrafusal stretch characteristics. InResearch in Muscle Development and the Muscle Spindle (ed. B. Q. Banker), pp. 299-332. Amsterdam:Excerpta Medica.

KARLSSON, U. L., HOOKER, W. M. & BENDEICH, E. (1971). Quantitative changes in the frog muscle spindle,with passive stretch. Journal of Ultrastructure Research 36, 743-756.

KATZ, B. (1961). The terminations of the afferent nerve fibre in the muscle spindle of the frog. PhilosophicalTransactions of the Royal Society of London, B 243, 221-240.

OVALLE, W. K. & SMITH, R. S. (1972). Histochemical identification of three types of intrafusal muscle fibersin the cat and monkey based on the myosin ATPase reaction. Canadian Journal of Physiology andPharmacology 50, 195-202.

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PAGE, S. G. (1966). Intrafusal muscle fibers in the frog. Journal of Microscopy 5, 101-104.SAITO, M., TOMONAGA, M. & NARABAYASHI, H. (1977). Histochemical study of normal muscle spindles.

Histochemical classification of intrafusal muscle fibers and intrafusal nerve endings. Journal of Neurology216, 79-89.

SMITH, R. S. (1963). Intrafusal musculature in Xenopus laevis. Acta physiologica scandinavica 59, Suppl.213.

STERLING, R. J. (1975). Muscle fibre types and collateral innervation in the frog spindle. Journal ofAnatomy119, 203-204.

YOSHIMURA, A., DIWAN, F. H., FUJITSUKA, N., SOKABE, M. & ITO, F. (1987). Classification of the intrafusalmuscle fibers in the frog muscle spindle. Journal of the Physiological Society of Japan 49, 477.