THE ANATOMICAL RECORD 226:168-176 (1990)
Variation in Histochemical Enzyme Profile and Diameter Along Human Masseter lntrafusal Muscle Fibers PER-OLOF ERIKSSON AND LARS-ERIC THORNELL Departments of Clinical Oral Physiology and Anatomy, University of Umeci, 901 87 Urneb, Sweden
,
ABSTRACT The histochemical enzyme profile of human masseter intrafusal muscle fibers was analyzed in consecutive serial cross sections along the individual fibers. Two hundred intrafusal fibers in 21 muscle spindles were classified. On the basis of equatorial nucleation, myosin ATPase-staining reactions after alkaline and acid preincubations and diameter, four different populations or types of intrafusal fiber were identified: large-diameter alkaline-stable and acid-stable fibers, bagz; two types of fiber with intermediate-diameter, alkaline-labile and acid-labile fibers corresponding t o bag, and alkaline-labile and acid-stable fibers designated as AS-bag,; and small-diameter alkaline-stable and acid-stable (pH 4.6)-acid-labile (pH 4.3) fibers called chain fibers. Regional variability in staining and diameter along the individual fibers was noted. In general, intrafusal fibers showed stronger oxidative reactions than did extrafusal fibers. The enzyme profile of the human masseter intrafusal fibers differed from that of extrafusal fibers in jaw, limb, and trunk muscles and also from that reported for spindles in limb and trunk muscles in man. The result suggests unique properties of human jaw muscle spindles and the jaw motor system. The sensory receptors embedded in a muscle are vitally important for nervous system integration required for coordinated movement. Although far outnumbered by extrafusal fibers, muscle receptors equal extrafusal fibers in amount of nervous traffic (Matthews, 1981). Muscle spindles are the intramuscular sensory receptors that signal information about the degree and velocity of stretch applied to a muscle; that is, they measure length (static response) and rate of change of length (dynamic response) (Boyd, 1980). A spindle consists of a bundle of specialized muscle fibers, the intrafusal fibers, surrounded by a connective-tissue capsule and runs parallel to the extrafusal muscle fibers (for review, see Barker and Banks, 1986). The human masseter muscle is involved in a number of significant activities such as mastication, postural control, speech, and emotional expression. Our previous enzyme histochemical studies have shown that the masseter has a complex extrafusal fiber-type composition different from that of limb and trunk muscles that suggests specialized functional demands (Eriksson, 1982; Eriksson and Thornell, 1983; Thornell et al., 1984; Butler-Browne et al., 1988). In addition, the masseter shows numerous unusually large and complexly arranged muscle spindles with a heterogeneous nonrandom distribution (Eriksson and Thornell, 1987). Extrafusal skeletal muscle fibers can be classified into different types on the basis of enzyme histochemical characteristics, which have been shown to correlate with physiological properties (for review, see Burke, 1981; Dubowitz, 1985; Schmalbruch, 1985). Thus, myofibrillar ATPase activity is correlated with 6 1990 WILEY-LISS. INC
speed of contraction and oxidative enzymes with resistance to fatigue. Although there is extensive literature from research and clinical diagnostics about the histochemical enzyme profile of human extrafusal muscle fibers (for review, see Dubowitz, 19851, there are only a few reports on the intrafusal enzyme histochemistry of human limb (Spiro and Beilin, 1969; Sahgal et al., 1976; Saito et al., 1977), trunk (Kucera and DoroviniZis, 1979), and jaw (Eriksson, 1982; Eriksson and Thornell, 1985; Eriksson et al., 1988) muscles. To attain further knowledge about muscle spindle structure and function and about mechanisms in jaw motor control, we have investigated the histochemical enzyme profile and the size of human masseter intrafusal muscle fibers in consecutive serial cross sections along the entire muscle spindle length. MATERIALS AND METHODS
Muscle specimen from the deep masseter muscle portion of a previously healthy male subject, age 24 years, with complete natural dentition, was obtained within 24 h post-mortem, a delay that does not hamper reliable histochemical fiber typing (Eriksson et al., 1980). The deep masseter portion was chosen a s it is known to contain numerous muscle spindles (Eriksson and Thornell, 1987). The investigation has been approved by The National Board of Health and Welfare, Stockholm, Sweden.
Received October 31, 1988; accepted March 7, 1989
HUMAN MASSETER INTRAFUSAL FIBER TYPES
Fig, 1. Serial cross sections of a deep masseter muscle spindle stained for the demonstration of myofibrillar ATPase at pH 9.4 (A), pH 4.6 (B), pH 4.3 (0,NADH-TR (D), and GT (E). Tracing (F) is for identification of bag1 fibers (3-51, bag, (11, chain (6-101, and AS-bag, ( 2 , l l ) . ~ 2 5 0 Bar . 50 +m.
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TABLE 1. No. intrafusal fibers of each fiber type
No. sections
Intrafusal fiber tvDe Bag1 AS-bag, Bag2 Chain Vari
No. fibers 63 22 33 74 8
Md 162 162 162 162 216
Min-Max
x
54-270 54-216 54-252 54-252 126-252
17.0 14.5 22.9 10.0 15.8
Diameter of intrafusal fiber, krn Range SD Min-Max x SD
%
2.7 3.0 3.0 3.0 2.9
40 40 50 56 60
10-23 10-21 18-28 5-20 12-20
6.8 5.8 11.5 5.6 9.5
3.6 3.3 3.9 3.0 3.8
No. staining sections (median value [Mdl and minimum and maximum nos. [Min-Maxl) on which examination of fibers were based. Diameter of fiber types; the mean value (3,the standard deviation (SD), and the min-max values were calculated from the median diameters of single fibers (see text). The variability of the diameter along the intrafusal fibers is expressed as the range, including the range in relation to the mean fiber diameter (%).
Chain
AS-Bagl pH
9.4 4.6 4.3
@ P"
@ 0 0
(3
15
10
0
0
Fig. 2. Human masseter intrafusal fiber types. Histochemical staining reactions for myofibrillar ATPase at alkaline and acid pH, and fiber diameter.
A muscle spindle consists of two poles extending from either side of the spindle equator. According to Banks et al. (19771, each spindle pole was arbitrarily divided into three regions: region A, extending from the center of the spindle equator to the end of the spindle periaxial fluid space; region B, encompassing the area from the termination of the periaxial fluid space to the end of the visible spindle capsule; and region C, corresponding to the extracapsular part of the spindle pole. The intrafusal fibers were classified on basis of morphological criteria, the equatorial distribution of nuclei and diameter, and the ATPase reaction after alkaline and acid preincubations (Barker and Banks, 1986). However, because of the presence of nuclei, the myofibrillar material in the equatorial regions is restricted to the thin peripheral rim of the fiber. Therefore, the equatorial part of the A-region was excluded from histochemical fiber-type classification due to weak or absent enzyme staining. Morphometric Analyses
Enzyme Histochemical Methods and Fiber-type Classification
After mounting for transverse sectioning in OCT compound (Tissue TekR, Miles Laboratories, Naperville, IL) the specimen was frozen in liquid propane chilled to -160°C with liquid nitrogen. Serial cross sections, 10-12 pm thick, were cut on a cryostat microtome at -2o"C, mounted on glass slides, and stained for the demonstration of myofibrillar Ca2 -activated adenosine triphoshatase (ATPase, EC 3.6.1.3) a t pH 9.4 (Padykula and Herman, 1955) after alkaline (pH 9.4) and acid (pH 4.6 and 4.3) preincubations (Brooke and Kaiser, 1970; Dubowitz, 1985) and for nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR, EC 1.6.99.3, a mitochondrial oxidative enzyme) (Scarpelli e t al., 1958) a s modified by Nystrom (1968). A modified Gomori trichrome staining (GT) (Engel and Cunningham, 1963) and a Verhoeff-van Gieson staining was used to distinguish spindle capsules, cell borders, and nuclear staining. Each series of staining contained three sections of every staining, that is, a total of 18 sections corresponding to a distance between series of about 200 pm. A total of 17 series of stained cross sections corresponding to about 3.4 mm of length were made and analyzed. Sections from the different levels were stained simultaneously for the same staining. The pattern of staining of all fibers could therefore be traced and compared along the length of each fiber. +
The intrafusal fiber diameter was measured with a caliper as the "lesser fiber diameter," that is, the maximum diameter across the narrowest aspect of the fiber (Dubowitz, 1985). The measurements were made on ATPase, pH 9.4, photographs at 250 times magnification and for every series of staining, that is, every 200 pm. From the measurements at different locations (series of staining) of the fibers, minimum, maximum, and median values for the diameter of each fiber were determined. From the median values of the fibers, mean fiber diameters of each fiber-type group (see below) were calculated. Analysis of variance was used to test the hypothesis of no difference in mean fiber diameter between different fiber types. RESULTS
Two hundred intrafusal fibers in 21 muscle spindles were examined in, on average, 162 (median value) sections (Table 1). On the basis of the equatorial nucle-
Fig. 3. Tracings of serial transverse sections of the same spindle as in Figures 1,4, and 5 . Nos. 1-13 indicate the level or series of sections, and nos. in parentheses, the distance between sections in pm. Intrafusal fibers have been labeled as in Figure 1;bag, fibers (3-5), bag, (l),chain (610),and AS-bag, (2,111, E is a n extrafusal fiber encompassed in the spindle capsule. N indicates nerve tissue.
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HUMAN MASSETER INTRAFUSAL FIBER TYPES
8 ( 1400)
1
9 (1600)
2 (200)
3 10
(400)
( 1800)
4 (600)
11 (2000)
5 (800)
6 ( 1000)
12 (2200)
7 (1200)
13
(2400)
Fig. 3.
P.-O. ERIKSSON AND L:E.
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ation, the myosin ATPase-staining reactions, and the fiber diameter, four different populations or types of fiber were detected. Fibers with nuclei concentrated to the equatorial region and varying degrees of light staining a t pH 9.4 and totally or almost totally inhibited staining reaction a t pH 4.6 and 4.3 (alkaline- and acid-labile reaction) were termed bag, fibers (Fig. 1, fibers 3-5; Fig. 2). Fibers with equatorial concentration of nuclei and varying degrees of light staining a t pH 9.4 (alkaline-labile reaction) and dark staining a t both pH 4.6 and 4.3 (acid-stable reaction) were termed acid-stable bag, fibers (AS-bag,) (Fig. 1, fibers 2 and 11; Fig. 2). Bag, and AS-bag, were of medium diameter (diameter f 17.0 and 14.5; SD 2.7 and 3.0 bm, respectively). Small diameter fibers (f 10; SD 3.0 Fm) with equatorial nuclei in a row and dark staining a t pH 9.4 (alkaline-stable reaction), dark staining at pH 4.6, and inhibited or almost inhibited (acid-labile) reaction a t pH 4.3 were called chain fibers (Fig. 1,fibers 6-10; Fig. 2). Large-diameter fibers (% 22.9; SD 3.0 pm) with a n equatorial concentration of nuclei and moderate to dark staining a t pH 9.4 (alkaline-stable reaction), dark staining a t pH 4.6, and dark to moderate staining a t pH 4.3 (acid-stable reaction) were termed bag, fibers (Fig. 1, fiber 1: Fig. 2). In addition, eight fibers from six muscle spindles (4% of the total fiber population; diameter f 15.8; SD 2.9 pm) showed extreme variability in ATPase staining, with complete transition from light to dark staining along the length of the fiber following both alkaline and acid perincubations (two fibers), alkaline preincubation only (two fibers), and acid preincubation only (four fibers). After staining for NADH-TR, all intrafusal fiber types showed varying degrees of moderate to very strong reactivity, which, in general, was stronger than that of the extrafusal muscle fibers that showed very weak to strong reaction (Fig. ID). In general, regional staining variability along the individual fibers (e.g., Fig. 4, fibers 2 and 11)was noted with all histochemical reactions employed. There was marked variability in diameter along fibers (Figs. 3-6). Mean fiber diameters, minimum and maximum values, and the range for each fiber-type group are summarized in Table 1. The relative variability (the range in relation to the mean fiber diameter) was 40% for the bag, and AS-bag, fibers, 50% for the bag,, and 56% for the chain fibers. The diameter of the bag, fibers was significantly the largest (P < ,001). The bag, fibers were significantly larger than were the AS-bagl and the chain fibers (P < .001). Finally, the AS-bagl diameter was larger than the chain-fiber diameter (P < .001). DISCUSSION
In this study of the human masseter intrafusal fibers, analyzing the consecutive serial cross sections along the length of each fiber, we have identified four different intrafusal fiber types; two types of medium diameter bag,, one large bag,, and one small diameter chain fiber. The result confirms and extends our findings in a previous study of the masseter, based on analyses of random muscle spindle cross sections (Eriksson and Thornell, 1985). In the present investigation, we have examined and taken into account also the vari-
THORNELL
ability in enzyme-profile and diameter along the individual fibers. This masseter intrafusal enzyme profile differs from that of the extrafusal muscle fibers in jaw muscles (Eriksson, 1982; Eriksson and Thornell 1983) and from limb muscles (Dubowitz, 1985) and also diverges from reported limb and trunk muscle spindle histochemistry in man (Spiro and Beilin, 1969; Sahgal et al., 1976; Saito et al., 1977; Kucera and Dorovini-Zis, 1979). On the basis of myosin ATPase activity, Spiro and Beilin (1969) described three types of intrafusal fiber in human limb muscles: two types of alkaline-labile nuclear bag fibers and one type of alkaline-stable nuclear chain fiber. Sahgal et al. (1976) also classified two types of bag fibers and one chain fiber type; however, all were alkaline stable. Saito et al. (1977) reported four intrafusal fiber types: alkaline-labile and alkaline-stable forms of both bag and chain fibers. Kucera and Dorovini-Zis (1979) identified three types of intrafusal fiber, two types of nuclear bag fibers; bag, (alkaline and acid labile), and larger bag, (alkaline stable-acid stable) and small diameter chain fibers that were alkaline stable and heterogeneously stained, lightly-moderatelydarkley, a t pH 4.3. This variation in typing of intrafusal muscle fibers may reflect differences in staining methodology. Our finding of four types of intrafusal fiber in the human masseter muscle could also be due to a unique intrafusal fiber-type profile for this muscle. Indeed, the human masseter extrafusal fibers differ in isomyosin composition from that of limb muscles (Butler-Browne et al., 1988) and the masseter intrafusal fibers show a complex expression of isomyosins (Eriksson et al., 1988). According to studies in human limb muscle intrafusal fibers, Spiro and Beilin (1969) and Saito et al. (1977) found the ATPase staining to be consistent along the entire fiber length except for the equatorial region where the myofibrillar material is restricted to the peripheral rim and the enzyme activity is thus weak. Kucera and Dorovini-Zis (1979) studied human external intercostal muscles and found regional variability in ATPase and NADH-TR staining along all intrafusal fiber types. Our present histochemical results support the observations of regional variations in enzymatic staining along individual intrafusal fibers described both in man (Spiro and Beilin, 1969; Saito et al., 1977; Kucera and Dorovini-Zis, 1979) and in other species (Yellin, 1974; Soukup, 1976; Kahn and Soukup, 1979, 1988). The variations we observed were less apparent for the chain fibers. Extreme variability in staining activity, for both alkaline and acid preincubations, was found in a few intrafusal fibers from six different muscle spindles. The variability of histochemical staining along the fiber length may reflect variability of isomyosin composition as has been shown by immunocytochemical methods for r a t limb muscle spindles (Maier et al., 1988; Pedrosa e t al., 1989). Similar studies on human masseter spindles are in progress in our laboratory (Eriksson et al., 1988; preliminary report). In staining for ATPase a t pH 9.4, extrafusal limb, trunk (Dubowitz, 19851, and jaw (Ringqvist, 1974; Eriksson, 1982; Eriksson and Thornell, 1983) muscle fibers are generally classified into type I fibers (lightly stained) and type I1 fibers (darkly stained). Following
HUMAN MASSETER INTRAFUSAL FIRER TYPES
Fig. 4. Serial cross sections of the same spindle as in Figures 1, 3, and 5 demonstrating enzyme staining and morphology at different locations or levels. Sections from series (i.e., level) 1 stained for GT (A) and ATPase, pH 9.4 (B); from series 2 ( a t 200 pm) stained for ATPase, pH 9.4 (C); series 4 (600 pm), pH 9.4 (D);series 7 (1,200 pm), pH 9.4 (E) and NADH-TR (F); series 11 (2,000 ym), pH 9.4 (G);series 12 (2,200 pm), pH 9.4 (H); series 13 (2,400),pH 9.4 (I) and NADH-TR
173
(K). Regional variation in staining after alkaline preincubation (pH 9.4) is seen in fibers 2 and 11 (for labeling see Figs. 1F and 3). Note two equatorial regions in this spindle complex; a t series 7 in the right group of fibers (E,F) and also in the parallel left group of intrafusal fibers a t series 13 (1,K). The concentration of nuclei in the “bag” regions is clearly visible (arrows in E,F,I,K).N indicates nerve tissue. x 160. Bar 50 pm.
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SERIES ------FIBRE1
6
2
3
4
5
6
7I
9I
8I
10 I 1 I1
1I2 13 I 14 I
Chain /
7
Chain
10
Chain
-
/
11
_ I
AS-Bagl
400
800
1200
1600
2000
2 4 0 0 pm
Fig. 5. Tracing of the intrafusal muscle fibers of the spindle in Figures 1, 3 , and 4 to illustrate the variability in fiber diameter at different levels (series). The number of series of sections (above), the
distance between different levels (below), and the fiber numbers are labeled as in Figures 1, 3, and 4. Vertical bar, diameter reference, 20
acid preincubations the type I1 fibers can be classified into subtypes. In contrast to limb and trunk muscles, human masticatory muscles show also a relatively large frequency of ATPase-intermediate fibers (staining in between that of type I and that of type I1 fibers at pH 9.41, which represent a part of the normal fiber population (Eriksson, 1982; Eriksson and Thornell, 1983). The extrafusal type I and ATPase-IM fibers stain dark after acid preincubations. In contrast to ex-
trafusal type I and ATPase-IM fibers, the bag, fibers were unstained after acid preincubations, whereas on the other hand, AS-bag, were acid stable, that is, similarly stained with type I and ATPase-IM. The small nuclear chain fibers were comparable in ATPase staining profile to the extrafusal type IIB. All intrafusal fiber types differed from extrafusal fiber types by showing stronger oxidative activity, a n observation in line with previous studies on human limb and trunk muscle
pm.
HUMAN MASSETER INTRAFUSAL FIBER TYPES
AS-Bag1
I
I
5 10 15 20 25 30 0 5 10 15 20 25 30
I
1
(17)
I
I
I
L
I
I
I
I
Chain
Bag2
0
I
175
I
I
(15)
I
I
I
I
I
I
-
I
I
I
!
-
I
1
1
1
I
1
0 5 10 15 20 25 30 3 5 4 0 0 5 10 15 20 25 30
(23)
(10) pm
Fig. 6. Illustration of the variability in fiber diameter along each of 200 intrafusal muscle fibers from 21 human masseter muscle spindles. Each line or dot represents one fiber showing (in pm)the range and the miminum and maximum values. Mean diameter (Wm) of each fiber-type group is given below in parentheses.
spindles (Spiro and Beilin, 1969; Sahgal e t al., 1976; Kucera and Dorovini-Zis, 1979). Although Sahgal et al. (1976) did not find any change in diameter or splitting of fibers in muscle spindles from normal limb muscles, Kucera and Dorovini-Zis (1979) described a variability in intrafusal fiber diameter in their material of human spindles. We report here that the fiber diameter of masseter intrafusal fibers may vary significantly along individual fibers, without the presence of fiber splitting (Desaki and Uehara, 1979). Fiber diameter alone on the single section can therefore not be used to differentiate reliably between nuclear bag and nuclear chain fibers. The same conclusion was made for rat limb muscles (Soukup, 1976).The diameter variation may imply significant differences also in functional properties along the fiber, although contraction or stretching probably contribute to the changes in fiber diameter. Recent studies by Edman et al. (1985)showed in frog extrafusal muscle fiber that the velocity of shortening varies along the length of the fiber and that each fiber may have a unique velocity pattern. This supports the view that kinetic properties of the myofilament system can differ from one region to another along the length of a muscle fiber.
integrated in the fusimotor neurones, undergoes the final adjustment in the muscle spindle before being linked to the skeletomotor neurones by the primary spindle afferents (Johansson, 1981). The role of muscle spindles as simple mechanoreceptors has therefore been revised (Johansson and Sojka, 1985). Evidence from studies on jaw muscle spindles in primates (Luschei and Goldberg, 1981) suggests separate roles in mandibular function for primary endings, known to enclose both bag and chain fibers, and secondary endings, of which the majority are located primarily on the nuclear chain fibers (Boyd, 1980; Barker and Banks, 1986). Primary endings are sensitive to small amplitude muscle-length changes and seem to respond to muscle stretch to help stabilize the mandible in locomotion, particularly in walking, running, and jumping. Secondary endings, on the other hand, seem to be less involved in the stretch reflex and serve to provide other central nervous system structures with information about muscle length and jaw position (Luschei and Goldberg, 1981).Correlations between enzyme histochemical and physiological properties (Burke, 1981; Schmalbruch, 1985) make it likely that the masseter intrafusal fibers differ also in mechanical and contractile properties. The special intrafusal enzyme activities and morphological features Functional Considerations demonstrated for the human masseter complies with Recent evidence suggests that there is a n intricate multiplicity in neural control. In conclusion, human masseter intrafusal muscle fimultisensory convergence from descending pathways and from muscle, skin, and joint afferents onto the static bers show a histochemical enzyme profile, which is difand dynamic gamma-motoneurones. The information, ferent from that of jaw and limb muscle extrafusal fi-
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bers and also from that previously reported for limb and trunk muscle spindles. The results imply unique properties of human jaw muscle spindles as part of a n intricate motor control system. ACKNOWLEDGMENTS
We thank Miss Inga Johansson and Miss Lena Soderberg for skillful technical assistance and Dr. Barbara Kay Grove for linguistic revision of the manuscript. Financial support was provided by the Swedish Medical Research Council (3934 and 68741, the K - 0 Hansson Foundation, Ume2 University, and the Swedish Dental Society. LITERATURE CITED Banks, R.W., D.W. Harker, and M.J. Stacey 1977 A study of mammalian intrafusal muscle fibres using a combined histochemical and ultrastructural technique. J. Anat., 123:783-796. Barker, D., and R.W. Banks 1986 The muscle spindle. In: Myology. A.G. Engel and B.Q. Banker, eds. McGraw-Hill, New York, Vol. 1, pp. 309-341. Boyd, IA 1980 The isolated mammalian muscle spindle. Trends Neurosci., 3:258-265. Brooke, M.H., and K.K. Kaiser 1970 Muscle fiber types: How many and what kind? Arch. Neurol. Psychiatr., 23:369-379. Burke, R.E. 1981 Motor units: Anatomy, physiology and functional organization. In: Handbook of Physiology, Section I. The Nervous System. Vol. 11. Motor Control. V.B. Brooks, ed. American Physiological Society, Bethesda, MD, pp. 345-422. Butler-Browne, G.S., P.-0. Eriksson, C. Laurent, and L.-E. Thornell 1988 Adult human masseter muscle fibers express myosin isozymes characteristic of development. Muscle Nerve, 11t610620. Desaki, J., and Y. Uehara 1979 Evidence of branching of intrafusal muscle fibres. J. Electron Microsc., 28t128-130. Dubowitz, V. 1985 Muscle Biopsy: A Practical Approach, Ed. 2. Bailliere Tindall, London. Edman, K.A.P., C. Reggiani, and G . Kronnie 1985 Differences in maximum velocity of shortening along single muscle fibres of the frog. J. Physiol., 365:147-163. Engel, W.K., and G.G. Cunningham 1963 Rapid examination of musl c l e tissue. An improved tr&hrome method for fresh-frozen biopsy sections. Neurology, 13t919-923. Eriksson, P.-0. 1982 Muscle fibre composition of the human mandibular locomotor system. Enzyme-histochemical and morphological characteristics of functionally different parts. Swed. Dent. J . Suppl 12:l-44. Eriksson, P.-O., and L.-E. Thornell 1983 Histochemical and morphological muscle fibre characteristics of the human masseter, the medial pterygoid and the temporal muscles. Arch. Oral Biol., 28: 781-795. Eriksson, P.-O., and L.-E. Thornell 1985 Heterogeneous intrafusal fibre composition of the human masseter muscle. In: The Mammalian Muscle Spindle. LA. Boyd and M.H. Gladden, eds, MacMillan Press, Hampshire, England, pp. 95-100. Epiksson, P.-O., and L.-E. Thornell 1987 Relation to extrafusal fibre type-composition in muscle-spindle structure and location in the human masseter muscle. Arch. Oral Biol., 32t483-491. Eriksson, P.-O., A. Eriksson, M. Ringqvist, and L.-E. Thornell 1980
The reliability of histochemical fibre typing of human necropsy muscles. Histochemistry, 65:193-205. Eriksson, P.-O., G.S. Butler-Browne, D.A. Fischman, B.K. Grove, S. Schiaffino, I. Virtanen, and L.-E. Thornell 1988 Myofibrillar and cytoskeletal proteins in human muscle spindles. In: Mechanoreceptors-Development, Structure and Function. P. Hnik, T. Soukup, R. Vejsada, and J . Zelena, eds. Plenum Press, New York, pp. 273-274. Johansson, H. 1981 Reflex control of gamma-motoneurones. Thesis, University of Umea, Umel, Sweden. Johansson, H., and P. Sojka 1985 Actions on gamma-motorneurons elicted by electrical stimulation of cutaneous afferent fibers in the hind limbs of the cat. J. Physiol., 366t343-363. Khan, M.A., and T. Soukup 1979 A histoenzymatic study of rat intrafusal muscle fibers. Histochemistry, 62t179-189. Khan, M.A., and T. Soukup 1988 Histochemical heterogeneity of intrafusal muscle fibres in slow and fast skeletal muscles of the rat. Histochem. J., 20:52-60. Kucera, J.,and K. Dorovini-Zis 1979 Types of human intrafusal muscle fibers. Muscle Nerve, 2:437-451. Luschei, E.S., and L.J. Goldberg 1981 Neural mechanisms of mandibular control: Mastication and voluntary biting. In: Handbook of Physiology, Section 1. The Nervous System. Vol. 11. Motor Control. V.B. Brooks, ed. American Physiological Society, Bethesda, MD, pp. 1237-1274. Maier, A., B. Gambke, and D. Pette 1988 Immunohistochemical demonstration of embryonic myosin heavy chains in adult mammalian intrafusal fibers. Histochemistry, 88:267-271. Matthews, P.B.C. 1981 Muscle spindles: Their messages and their fusiomotor supply. In: Handbook of Physiology, Section 1. The Nervous System. Vol. 11. Motor Control. V.B. Brooks, ed. American Physiological Society, Bethesda, MD, pp. 189-228. Nystrom, B. 1968 Histochemistry of developing cat muscles. Acta Neurol. Scand., 44:405-439. Padykula, H.A., and E. Herman 1955 The specificity of the histochemical method for adenosine triphosphatase. J . Histochem. Cytochem., 3:170-183. Pedrosa, F., G.S. Butler-Browne, G.K. Dhoot, D.A. Fischman, and L.-E. Thornell 1989 Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles. Histochemistry, 92Ain press). Ringqvist, M. 1974 Fiber types in human masticatory muscles. Relation to function. Scand. J. Dent. Res., 82t333-355. Sahgal, V., C.A. Morgen, and S.Sahgal 1976 Histochemical and morphological changes in human muscle spindle in upper and lower motor neuron lesions. Acta Neuropathol., 34:41-46. Saito, M., M. Tomonaga, K. Hirayama, and H. Narabayshi 1977 Histochemical study of normal human muscle spindle. J . Neurol., 216r79-89. Scarpelli, D.G., R. Hess, and A.G.E. Pearse 1958 The cytochemical localization of oxidative enzymes. J. Biophys. Biochem. Cytol., 4347-751. Schmalbruch, H. 1985 Skeletal muscle. In: Handbook of Microscopic Anatomy, Part 6, Vol. 2. A. Oksche and L. Vollrath, eds. Springer, Berlin. Soukup, T. 1976 Intrafusal fibre types in rat limb muscle spindles. Histochemistrv, 47t43 -57. Spiro, A.J., and R.L. Beilin 1969 Human muscle spindle histochemistry. Arch. Neurol., 20.271-275. Thornell, L.-E., R. Billeter, P.-0. Eriksson, and M. Ringavist 1984 Heterogeneous distribution of myosin i n human masticatory muscle fibres a s shown by immunocytochemistry. Arch. Oral Biol.. 29:l-5. Yellin, H. 1974 Regional differences in the contractile apparatus of intrafusal muscle fibres. Am. J. Anat., 139:147-152.
Variation in Histochemical Enzyme Profile and Diameter Along Human Masseter lntrafusal Muscle Fibers PER-OLOF ERIKSSON AND LARS-ERIC THORNELL Departments of Clinical Oral Physiology and Anatomy, University of Umeci, 901 87 Urneb, Sweden
,
ABSTRACT The histochemical enzyme profile of human masseter intrafusal muscle fibers was analyzed in consecutive serial cross sections along the individual fibers. Two hundred intrafusal fibers in 21 muscle spindles were classified. On the basis of equatorial nucleation, myosin ATPase-staining reactions after alkaline and acid preincubations and diameter, four different populations or types of intrafusal fiber were identified: large-diameter alkaline-stable and acid-stable fibers, bagz; two types of fiber with intermediate-diameter, alkaline-labile and acid-labile fibers corresponding t o bag, and alkaline-labile and acid-stable fibers designated as AS-bag,; and small-diameter alkaline-stable and acid-stable (pH 4.6)-acid-labile (pH 4.3) fibers called chain fibers. Regional variability in staining and diameter along the individual fibers was noted. In general, intrafusal fibers showed stronger oxidative reactions than did extrafusal fibers. The enzyme profile of the human masseter intrafusal fibers differed from that of extrafusal fibers in jaw, limb, and trunk muscles and also from that reported for spindles in limb and trunk muscles in man. The result suggests unique properties of human jaw muscle spindles and the jaw motor system. The sensory receptors embedded in a muscle are vitally important for nervous system integration required for coordinated movement. Although far outnumbered by extrafusal fibers, muscle receptors equal extrafusal fibers in amount of nervous traffic (Matthews, 1981). Muscle spindles are the intramuscular sensory receptors that signal information about the degree and velocity of stretch applied to a muscle; that is, they measure length (static response) and rate of change of length (dynamic response) (Boyd, 1980). A spindle consists of a bundle of specialized muscle fibers, the intrafusal fibers, surrounded by a connective-tissue capsule and runs parallel to the extrafusal muscle fibers (for review, see Barker and Banks, 1986). The human masseter muscle is involved in a number of significant activities such as mastication, postural control, speech, and emotional expression. Our previous enzyme histochemical studies have shown that the masseter has a complex extrafusal fiber-type composition different from that of limb and trunk muscles that suggests specialized functional demands (Eriksson, 1982; Eriksson and Thornell, 1983; Thornell et al., 1984; Butler-Browne et al., 1988). In addition, the masseter shows numerous unusually large and complexly arranged muscle spindles with a heterogeneous nonrandom distribution (Eriksson and Thornell, 1987). Extrafusal skeletal muscle fibers can be classified into different types on the basis of enzyme histochemical characteristics, which have been shown to correlate with physiological properties (for review, see Burke, 1981; Dubowitz, 1985; Schmalbruch, 1985). Thus, myofibrillar ATPase activity is correlated with 6 1990 WILEY-LISS. INC
speed of contraction and oxidative enzymes with resistance to fatigue. Although there is extensive literature from research and clinical diagnostics about the histochemical enzyme profile of human extrafusal muscle fibers (for review, see Dubowitz, 19851, there are only a few reports on the intrafusal enzyme histochemistry of human limb (Spiro and Beilin, 1969; Sahgal et al., 1976; Saito et al., 1977), trunk (Kucera and DoroviniZis, 1979), and jaw (Eriksson, 1982; Eriksson and Thornell, 1985; Eriksson et al., 1988) muscles. To attain further knowledge about muscle spindle structure and function and about mechanisms in jaw motor control, we have investigated the histochemical enzyme profile and the size of human masseter intrafusal muscle fibers in consecutive serial cross sections along the entire muscle spindle length. MATERIALS AND METHODS
Muscle specimen from the deep masseter muscle portion of a previously healthy male subject, age 24 years, with complete natural dentition, was obtained within 24 h post-mortem, a delay that does not hamper reliable histochemical fiber typing (Eriksson et al., 1980). The deep masseter portion was chosen a s it is known to contain numerous muscle spindles (Eriksson and Thornell, 1987). The investigation has been approved by The National Board of Health and Welfare, Stockholm, Sweden.
Received October 31, 1988; accepted March 7, 1989
HUMAN MASSETER INTRAFUSAL FIBER TYPES
Fig, 1. Serial cross sections of a deep masseter muscle spindle stained for the demonstration of myofibrillar ATPase at pH 9.4 (A), pH 4.6 (B), pH 4.3 (0,NADH-TR (D), and GT (E). Tracing (F) is for identification of bag1 fibers (3-51, bag, (11, chain (6-101, and AS-bag, ( 2 , l l ) . ~ 2 5 0 Bar . 50 +m.
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TABLE 1. No. intrafusal fibers of each fiber type
No. sections
Intrafusal fiber tvDe Bag1 AS-bag, Bag2 Chain Vari
No. fibers 63 22 33 74 8
Md 162 162 162 162 216
Min-Max
x
54-270 54-216 54-252 54-252 126-252
17.0 14.5 22.9 10.0 15.8
Diameter of intrafusal fiber, krn Range SD Min-Max x SD
%
2.7 3.0 3.0 3.0 2.9
40 40 50 56 60
10-23 10-21 18-28 5-20 12-20
6.8 5.8 11.5 5.6 9.5
3.6 3.3 3.9 3.0 3.8
No. staining sections (median value [Mdl and minimum and maximum nos. [Min-Maxl) on which examination of fibers were based. Diameter of fiber types; the mean value (3,the standard deviation (SD), and the min-max values were calculated from the median diameters of single fibers (see text). The variability of the diameter along the intrafusal fibers is expressed as the range, including the range in relation to the mean fiber diameter (%).
Chain
AS-Bagl pH
9.4 4.6 4.3
@ P"
@ 0 0
(3
15
10
0
0
Fig. 2. Human masseter intrafusal fiber types. Histochemical staining reactions for myofibrillar ATPase at alkaline and acid pH, and fiber diameter.
A muscle spindle consists of two poles extending from either side of the spindle equator. According to Banks et al. (19771, each spindle pole was arbitrarily divided into three regions: region A, extending from the center of the spindle equator to the end of the spindle periaxial fluid space; region B, encompassing the area from the termination of the periaxial fluid space to the end of the visible spindle capsule; and region C, corresponding to the extracapsular part of the spindle pole. The intrafusal fibers were classified on basis of morphological criteria, the equatorial distribution of nuclei and diameter, and the ATPase reaction after alkaline and acid preincubations (Barker and Banks, 1986). However, because of the presence of nuclei, the myofibrillar material in the equatorial regions is restricted to the thin peripheral rim of the fiber. Therefore, the equatorial part of the A-region was excluded from histochemical fiber-type classification due to weak or absent enzyme staining. Morphometric Analyses
Enzyme Histochemical Methods and Fiber-type Classification
After mounting for transverse sectioning in OCT compound (Tissue TekR, Miles Laboratories, Naperville, IL) the specimen was frozen in liquid propane chilled to -160°C with liquid nitrogen. Serial cross sections, 10-12 pm thick, were cut on a cryostat microtome at -2o"C, mounted on glass slides, and stained for the demonstration of myofibrillar Ca2 -activated adenosine triphoshatase (ATPase, EC 3.6.1.3) a t pH 9.4 (Padykula and Herman, 1955) after alkaline (pH 9.4) and acid (pH 4.6 and 4.3) preincubations (Brooke and Kaiser, 1970; Dubowitz, 1985) and for nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR, EC 1.6.99.3, a mitochondrial oxidative enzyme) (Scarpelli e t al., 1958) a s modified by Nystrom (1968). A modified Gomori trichrome staining (GT) (Engel and Cunningham, 1963) and a Verhoeff-van Gieson staining was used to distinguish spindle capsules, cell borders, and nuclear staining. Each series of staining contained three sections of every staining, that is, a total of 18 sections corresponding to a distance between series of about 200 pm. A total of 17 series of stained cross sections corresponding to about 3.4 mm of length were made and analyzed. Sections from the different levels were stained simultaneously for the same staining. The pattern of staining of all fibers could therefore be traced and compared along the length of each fiber. +
The intrafusal fiber diameter was measured with a caliper as the "lesser fiber diameter," that is, the maximum diameter across the narrowest aspect of the fiber (Dubowitz, 1985). The measurements were made on ATPase, pH 9.4, photographs at 250 times magnification and for every series of staining, that is, every 200 pm. From the measurements at different locations (series of staining) of the fibers, minimum, maximum, and median values for the diameter of each fiber were determined. From the median values of the fibers, mean fiber diameters of each fiber-type group (see below) were calculated. Analysis of variance was used to test the hypothesis of no difference in mean fiber diameter between different fiber types. RESULTS
Two hundred intrafusal fibers in 21 muscle spindles were examined in, on average, 162 (median value) sections (Table 1). On the basis of the equatorial nucle-
Fig. 3. Tracings of serial transverse sections of the same spindle as in Figures 1,4, and 5 . Nos. 1-13 indicate the level or series of sections, and nos. in parentheses, the distance between sections in pm. Intrafusal fibers have been labeled as in Figure 1;bag, fibers (3-5), bag, (l),chain (610),and AS-bag, (2,111, E is a n extrafusal fiber encompassed in the spindle capsule. N indicates nerve tissue.
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HUMAN MASSETER INTRAFUSAL FIBER TYPES
8 ( 1400)
1
9 (1600)
2 (200)
3 10
(400)
( 1800)
4 (600)
11 (2000)
5 (800)
6 ( 1000)
12 (2200)
7 (1200)
13
(2400)
Fig. 3.
P.-O. ERIKSSON AND L:E.
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ation, the myosin ATPase-staining reactions, and the fiber diameter, four different populations or types of fiber were detected. Fibers with nuclei concentrated to the equatorial region and varying degrees of light staining a t pH 9.4 and totally or almost totally inhibited staining reaction a t pH 4.6 and 4.3 (alkaline- and acid-labile reaction) were termed bag, fibers (Fig. 1, fibers 3-5; Fig. 2). Fibers with equatorial concentration of nuclei and varying degrees of light staining a t pH 9.4 (alkaline-labile reaction) and dark staining a t both pH 4.6 and 4.3 (acid-stable reaction) were termed acid-stable bag, fibers (AS-bag,) (Fig. 1, fibers 2 and 11; Fig. 2). Bag, and AS-bag, were of medium diameter (diameter f 17.0 and 14.5; SD 2.7 and 3.0 bm, respectively). Small diameter fibers (f 10; SD 3.0 Fm) with equatorial nuclei in a row and dark staining a t pH 9.4 (alkaline-stable reaction), dark staining at pH 4.6, and inhibited or almost inhibited (acid-labile) reaction a t pH 4.3 were called chain fibers (Fig. 1,fibers 6-10; Fig. 2). Large-diameter fibers (% 22.9; SD 3.0 pm) with a n equatorial concentration of nuclei and moderate to dark staining a t pH 9.4 (alkaline-stable reaction), dark staining a t pH 4.6, and dark to moderate staining a t pH 4.3 (acid-stable reaction) were termed bag, fibers (Fig. 1, fiber 1: Fig. 2). In addition, eight fibers from six muscle spindles (4% of the total fiber population; diameter f 15.8; SD 2.9 pm) showed extreme variability in ATPase staining, with complete transition from light to dark staining along the length of the fiber following both alkaline and acid perincubations (two fibers), alkaline preincubation only (two fibers), and acid preincubation only (four fibers). After staining for NADH-TR, all intrafusal fiber types showed varying degrees of moderate to very strong reactivity, which, in general, was stronger than that of the extrafusal muscle fibers that showed very weak to strong reaction (Fig. ID). In general, regional staining variability along the individual fibers (e.g., Fig. 4, fibers 2 and 11)was noted with all histochemical reactions employed. There was marked variability in diameter along fibers (Figs. 3-6). Mean fiber diameters, minimum and maximum values, and the range for each fiber-type group are summarized in Table 1. The relative variability (the range in relation to the mean fiber diameter) was 40% for the bag, and AS-bag, fibers, 50% for the bag,, and 56% for the chain fibers. The diameter of the bag, fibers was significantly the largest (P < ,001). The bag, fibers were significantly larger than were the AS-bagl and the chain fibers (P < .001). Finally, the AS-bagl diameter was larger than the chain-fiber diameter (P < .001). DISCUSSION
In this study of the human masseter intrafusal fibers, analyzing the consecutive serial cross sections along the length of each fiber, we have identified four different intrafusal fiber types; two types of medium diameter bag,, one large bag,, and one small diameter chain fiber. The result confirms and extends our findings in a previous study of the masseter, based on analyses of random muscle spindle cross sections (Eriksson and Thornell, 1985). In the present investigation, we have examined and taken into account also the vari-
THORNELL
ability in enzyme-profile and diameter along the individual fibers. This masseter intrafusal enzyme profile differs from that of the extrafusal muscle fibers in jaw muscles (Eriksson, 1982; Eriksson and Thornell 1983) and from limb muscles (Dubowitz, 1985) and also diverges from reported limb and trunk muscle spindle histochemistry in man (Spiro and Beilin, 1969; Sahgal et al., 1976; Saito et al., 1977; Kucera and Dorovini-Zis, 1979). On the basis of myosin ATPase activity, Spiro and Beilin (1969) described three types of intrafusal fiber in human limb muscles: two types of alkaline-labile nuclear bag fibers and one type of alkaline-stable nuclear chain fiber. Sahgal et al. (1976) also classified two types of bag fibers and one chain fiber type; however, all were alkaline stable. Saito et al. (1977) reported four intrafusal fiber types: alkaline-labile and alkaline-stable forms of both bag and chain fibers. Kucera and Dorovini-Zis (1979) identified three types of intrafusal fiber, two types of nuclear bag fibers; bag, (alkaline and acid labile), and larger bag, (alkaline stable-acid stable) and small diameter chain fibers that were alkaline stable and heterogeneously stained, lightly-moderatelydarkley, a t pH 4.3. This variation in typing of intrafusal muscle fibers may reflect differences in staining methodology. Our finding of four types of intrafusal fiber in the human masseter muscle could also be due to a unique intrafusal fiber-type profile for this muscle. Indeed, the human masseter extrafusal fibers differ in isomyosin composition from that of limb muscles (Butler-Browne et al., 1988) and the masseter intrafusal fibers show a complex expression of isomyosins (Eriksson et al., 1988). According to studies in human limb muscle intrafusal fibers, Spiro and Beilin (1969) and Saito et al. (1977) found the ATPase staining to be consistent along the entire fiber length except for the equatorial region where the myofibrillar material is restricted to the peripheral rim and the enzyme activity is thus weak. Kucera and Dorovini-Zis (1979) studied human external intercostal muscles and found regional variability in ATPase and NADH-TR staining along all intrafusal fiber types. Our present histochemical results support the observations of regional variations in enzymatic staining along individual intrafusal fibers described both in man (Spiro and Beilin, 1969; Saito et al., 1977; Kucera and Dorovini-Zis, 1979) and in other species (Yellin, 1974; Soukup, 1976; Kahn and Soukup, 1979, 1988). The variations we observed were less apparent for the chain fibers. Extreme variability in staining activity, for both alkaline and acid preincubations, was found in a few intrafusal fibers from six different muscle spindles. The variability of histochemical staining along the fiber length may reflect variability of isomyosin composition as has been shown by immunocytochemical methods for r a t limb muscle spindles (Maier et al., 1988; Pedrosa e t al., 1989). Similar studies on human masseter spindles are in progress in our laboratory (Eriksson et al., 1988; preliminary report). In staining for ATPase a t pH 9.4, extrafusal limb, trunk (Dubowitz, 19851, and jaw (Ringqvist, 1974; Eriksson, 1982; Eriksson and Thornell, 1983) muscle fibers are generally classified into type I fibers (lightly stained) and type I1 fibers (darkly stained). Following
HUMAN MASSETER INTRAFUSAL FIRER TYPES
Fig. 4. Serial cross sections of the same spindle as in Figures 1, 3, and 5 demonstrating enzyme staining and morphology at different locations or levels. Sections from series (i.e., level) 1 stained for GT (A) and ATPase, pH 9.4 (B); from series 2 ( a t 200 pm) stained for ATPase, pH 9.4 (C); series 4 (600 pm), pH 9.4 (D);series 7 (1,200 pm), pH 9.4 (E) and NADH-TR (F); series 11 (2,000 ym), pH 9.4 (G);series 12 (2,200 pm), pH 9.4 (H); series 13 (2,400),pH 9.4 (I) and NADH-TR
173
(K). Regional variation in staining after alkaline preincubation (pH 9.4) is seen in fibers 2 and 11 (for labeling see Figs. 1F and 3). Note two equatorial regions in this spindle complex; a t series 7 in the right group of fibers (E,F) and also in the parallel left group of intrafusal fibers a t series 13 (1,K). The concentration of nuclei in the “bag” regions is clearly visible (arrows in E,F,I,K).N indicates nerve tissue. x 160. Bar 50 pm.
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SERIES ------FIBRE1
6
2
3
4
5
6
7I
9I
8I
10 I 1 I1
1I2 13 I 14 I
Chain /
7
Chain
10
Chain
-
/
11
_ I
AS-Bagl
400
800
1200
1600
2000
2 4 0 0 pm
Fig. 5. Tracing of the intrafusal muscle fibers of the spindle in Figures 1, 3 , and 4 to illustrate the variability in fiber diameter at different levels (series). The number of series of sections (above), the
distance between different levels (below), and the fiber numbers are labeled as in Figures 1, 3, and 4. Vertical bar, diameter reference, 20
acid preincubations the type I1 fibers can be classified into subtypes. In contrast to limb and trunk muscles, human masticatory muscles show also a relatively large frequency of ATPase-intermediate fibers (staining in between that of type I and that of type I1 fibers at pH 9.41, which represent a part of the normal fiber population (Eriksson, 1982; Eriksson and Thornell, 1983). The extrafusal type I and ATPase-IM fibers stain dark after acid preincubations. In contrast to ex-
trafusal type I and ATPase-IM fibers, the bag, fibers were unstained after acid preincubations, whereas on the other hand, AS-bag, were acid stable, that is, similarly stained with type I and ATPase-IM. The small nuclear chain fibers were comparable in ATPase staining profile to the extrafusal type IIB. All intrafusal fiber types differed from extrafusal fiber types by showing stronger oxidative activity, a n observation in line with previous studies on human limb and trunk muscle
pm.
HUMAN MASSETER INTRAFUSAL FIBER TYPES
AS-Bag1
I
I
5 10 15 20 25 30 0 5 10 15 20 25 30
I
1
(17)
I
I
I
L
I
I
I
I
Chain
Bag2
0
I
175
I
I
(15)
I
I
I
I
I
I
-
I
I
I
!
-
I
1
1
1
I
1
0 5 10 15 20 25 30 3 5 4 0 0 5 10 15 20 25 30
(23)
(10) pm
Fig. 6. Illustration of the variability in fiber diameter along each of 200 intrafusal muscle fibers from 21 human masseter muscle spindles. Each line or dot represents one fiber showing (in pm)the range and the miminum and maximum values. Mean diameter (Wm) of each fiber-type group is given below in parentheses.
spindles (Spiro and Beilin, 1969; Sahgal e t al., 1976; Kucera and Dorovini-Zis, 1979). Although Sahgal et al. (1976) did not find any change in diameter or splitting of fibers in muscle spindles from normal limb muscles, Kucera and Dorovini-Zis (1979) described a variability in intrafusal fiber diameter in their material of human spindles. We report here that the fiber diameter of masseter intrafusal fibers may vary significantly along individual fibers, without the presence of fiber splitting (Desaki and Uehara, 1979). Fiber diameter alone on the single section can therefore not be used to differentiate reliably between nuclear bag and nuclear chain fibers. The same conclusion was made for rat limb muscles (Soukup, 1976).The diameter variation may imply significant differences also in functional properties along the fiber, although contraction or stretching probably contribute to the changes in fiber diameter. Recent studies by Edman et al. (1985)showed in frog extrafusal muscle fiber that the velocity of shortening varies along the length of the fiber and that each fiber may have a unique velocity pattern. This supports the view that kinetic properties of the myofilament system can differ from one region to another along the length of a muscle fiber.
integrated in the fusimotor neurones, undergoes the final adjustment in the muscle spindle before being linked to the skeletomotor neurones by the primary spindle afferents (Johansson, 1981). The role of muscle spindles as simple mechanoreceptors has therefore been revised (Johansson and Sojka, 1985). Evidence from studies on jaw muscle spindles in primates (Luschei and Goldberg, 1981) suggests separate roles in mandibular function for primary endings, known to enclose both bag and chain fibers, and secondary endings, of which the majority are located primarily on the nuclear chain fibers (Boyd, 1980; Barker and Banks, 1986). Primary endings are sensitive to small amplitude muscle-length changes and seem to respond to muscle stretch to help stabilize the mandible in locomotion, particularly in walking, running, and jumping. Secondary endings, on the other hand, seem to be less involved in the stretch reflex and serve to provide other central nervous system structures with information about muscle length and jaw position (Luschei and Goldberg, 1981).Correlations between enzyme histochemical and physiological properties (Burke, 1981; Schmalbruch, 1985) make it likely that the masseter intrafusal fibers differ also in mechanical and contractile properties. The special intrafusal enzyme activities and morphological features Functional Considerations demonstrated for the human masseter complies with Recent evidence suggests that there is a n intricate multiplicity in neural control. In conclusion, human masseter intrafusal muscle fimultisensory convergence from descending pathways and from muscle, skin, and joint afferents onto the static bers show a histochemical enzyme profile, which is difand dynamic gamma-motoneurones. The information, ferent from that of jaw and limb muscle extrafusal fi-
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bers and also from that previously reported for limb and trunk muscle spindles. The results imply unique properties of human jaw muscle spindles as part of a n intricate motor control system. ACKNOWLEDGMENTS
We thank Miss Inga Johansson and Miss Lena Soderberg for skillful technical assistance and Dr. Barbara Kay Grove for linguistic revision of the manuscript. Financial support was provided by the Swedish Medical Research Council (3934 and 68741, the K - 0 Hansson Foundation, Ume2 University, and the Swedish Dental Society. LITERATURE CITED Banks, R.W., D.W. Harker, and M.J. Stacey 1977 A study of mammalian intrafusal muscle fibres using a combined histochemical and ultrastructural technique. J. Anat., 123:783-796. Barker, D., and R.W. Banks 1986 The muscle spindle. In: Myology. A.G. Engel and B.Q. Banker, eds. McGraw-Hill, New York, Vol. 1, pp. 309-341. Boyd, IA 1980 The isolated mammalian muscle spindle. Trends Neurosci., 3:258-265. Brooke, M.H., and K.K. Kaiser 1970 Muscle fiber types: How many and what kind? Arch. Neurol. Psychiatr., 23:369-379. Burke, R.E. 1981 Motor units: Anatomy, physiology and functional organization. In: Handbook of Physiology, Section I. The Nervous System. Vol. 11. Motor Control. V.B. Brooks, ed. American Physiological Society, Bethesda, MD, pp. 345-422. Butler-Browne, G.S., P.-0. Eriksson, C. Laurent, and L.-E. Thornell 1988 Adult human masseter muscle fibers express myosin isozymes characteristic of development. Muscle Nerve, 11t610620. Desaki, J., and Y. Uehara 1979 Evidence of branching of intrafusal muscle fibres. J. Electron Microsc., 28t128-130. Dubowitz, V. 1985 Muscle Biopsy: A Practical Approach, Ed. 2. Bailliere Tindall, London. Edman, K.A.P., C. Reggiani, and G . Kronnie 1985 Differences in maximum velocity of shortening along single muscle fibres of the frog. J. Physiol., 365:147-163. Engel, W.K., and G.G. Cunningham 1963 Rapid examination of musl c l e tissue. An improved tr&hrome method for fresh-frozen biopsy sections. Neurology, 13t919-923. Eriksson, P.-0. 1982 Muscle fibre composition of the human mandibular locomotor system. Enzyme-histochemical and morphological characteristics of functionally different parts. Swed. Dent. J . Suppl 12:l-44. Eriksson, P.-O., and L.-E. Thornell 1983 Histochemical and morphological muscle fibre characteristics of the human masseter, the medial pterygoid and the temporal muscles. Arch. Oral Biol., 28: 781-795. Eriksson, P.-O., and L.-E. Thornell 1985 Heterogeneous intrafusal fibre composition of the human masseter muscle. In: The Mammalian Muscle Spindle. LA. Boyd and M.H. Gladden, eds, MacMillan Press, Hampshire, England, pp. 95-100. Epiksson, P.-O., and L.-E. Thornell 1987 Relation to extrafusal fibre type-composition in muscle-spindle structure and location in the human masseter muscle. Arch. Oral Biol., 32t483-491. Eriksson, P.-O., A. Eriksson, M. Ringqvist, and L.-E. Thornell 1980
The reliability of histochemical fibre typing of human necropsy muscles. Histochemistry, 65:193-205. Eriksson, P.-O., G.S. Butler-Browne, D.A. Fischman, B.K. Grove, S. Schiaffino, I. Virtanen, and L.-E. Thornell 1988 Myofibrillar and cytoskeletal proteins in human muscle spindles. In: Mechanoreceptors-Development, Structure and Function. P. Hnik, T. Soukup, R. Vejsada, and J . Zelena, eds. Plenum Press, New York, pp. 273-274. Johansson, H. 1981 Reflex control of gamma-motoneurones. Thesis, University of Umea, Umel, Sweden. Johansson, H., and P. Sojka 1985 Actions on gamma-motorneurons elicted by electrical stimulation of cutaneous afferent fibers in the hind limbs of the cat. J. Physiol., 366t343-363. Khan, M.A., and T. Soukup 1979 A histoenzymatic study of rat intrafusal muscle fibers. Histochemistry, 62t179-189. Khan, M.A., and T. Soukup 1988 Histochemical heterogeneity of intrafusal muscle fibres in slow and fast skeletal muscles of the rat. Histochem. J., 20:52-60. Kucera, J.,and K. Dorovini-Zis 1979 Types of human intrafusal muscle fibers. Muscle Nerve, 2:437-451. Luschei, E.S., and L.J. Goldberg 1981 Neural mechanisms of mandibular control: Mastication and voluntary biting. In: Handbook of Physiology, Section 1. The Nervous System. Vol. 11. Motor Control. V.B. Brooks, ed. American Physiological Society, Bethesda, MD, pp. 1237-1274. Maier, A., B. Gambke, and D. Pette 1988 Immunohistochemical demonstration of embryonic myosin heavy chains in adult mammalian intrafusal fibers. Histochemistry, 88:267-271. Matthews, P.B.C. 1981 Muscle spindles: Their messages and their fusiomotor supply. In: Handbook of Physiology, Section 1. The Nervous System. Vol. 11. Motor Control. V.B. Brooks, ed. American Physiological Society, Bethesda, MD, pp. 189-228. Nystrom, B. 1968 Histochemistry of developing cat muscles. Acta Neurol. Scand., 44:405-439. Padykula, H.A., and E. Herman 1955 The specificity of the histochemical method for adenosine triphosphatase. J . Histochem. Cytochem., 3:170-183. Pedrosa, F., G.S. Butler-Browne, G.K. Dhoot, D.A. Fischman, and L.-E. Thornell 1989 Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles. Histochemistry, 92Ain press). Ringqvist, M. 1974 Fiber types in human masticatory muscles. Relation to function. Scand. J. Dent. Res., 82t333-355. Sahgal, V., C.A. Morgen, and S.Sahgal 1976 Histochemical and morphological changes in human muscle spindle in upper and lower motor neuron lesions. Acta Neuropathol., 34:41-46. Saito, M., M. Tomonaga, K. Hirayama, and H. Narabayshi 1977 Histochemical study of normal human muscle spindle. J . Neurol., 216r79-89. Scarpelli, D.G., R. Hess, and A.G.E. Pearse 1958 The cytochemical localization of oxidative enzymes. J. Biophys. Biochem. Cytol., 4347-751. Schmalbruch, H. 1985 Skeletal muscle. In: Handbook of Microscopic Anatomy, Part 6, Vol. 2. A. Oksche and L. Vollrath, eds. Springer, Berlin. Soukup, T. 1976 Intrafusal fibre types in rat limb muscle spindles. Histochemistrv, 47t43 -57. Spiro, A.J., and R.L. Beilin 1969 Human muscle spindle histochemistry. Arch. Neurol., 20.271-275. Thornell, L.-E., R. Billeter, P.-0. Eriksson, and M. Ringavist 1984 Heterogeneous distribution of myosin i n human masticatory muscle fibres a s shown by immunocytochemistry. Arch. Oral Biol.. 29:l-5. Yellin, H. 1974 Regional differences in the contractile apparatus of intrafusal muscle fibres. Am. J. Anat., 139:147-152.
