Muscle Spindle Composition and Distribution in Human Young Masseter and Biceps Brachii Muscles Reveal Early Growth and Maturation.код для вставкиСкачать
THE ANATOMICAL RECORD 294:683–693 (2011) Muscle Spindle Composition and Distribution in Human Young Masseter and Biceps Brachii Muscles Reveal Early Growth and Maturation CATHARINA ÖSTERLUND,1 JING-XIA LIU,2 LARS-ERIC THORNELL,2 1 AND PER-OLOF ERIKSSON * 1 Department of Odontology, Clinical Oral Physiology, Umeå University, Umeå, Sweden 2 Department of Integrative Medical Biology, Section of Anatomy, Umeå University, Umeå, Sweden ABSTRACT Signiﬁcant changes in extrafusal ﬁber type composition take place in the human masseter muscle from young age, 3–7 years, to adulthood, in parallel with jaw-face skeleton growth, changes of dentitions and improvement of jaw functions. As motor and sensory control systems of muscles are interlinked, also the intrafusal ﬁber population, that is, muscle spindles, should undergo age-related changes in ﬁber type appearance. To test this hypothesis, we examined muscle spindles in the young masseter muscle and compared the result with previous data on adult masseter spindles. Also muscle spindles in the young biceps brachii muscle were examined. The result showed that muscle spindle composition and distribution were alike in young and adult masseter. As for the adult masseter, young masseter contained exceptionally large muscle spindles, and with the highest spindle density and most complex spindles found in the deep masseter portion. Hence, contrary to our hypothesis, masseter spindles do not undergo major morphological changes between young age and adulthood. Also in the biceps, young spindles were alike adult spindles. Taken together, the results showed that human masseter and biceps muscle spindles are morphologically mature already at young age. We conclude that muscle spindles in the human young masseter and biceps precede the extrafusal ﬁber population in growth and maturation. This in turn suggests early reﬂex control and proprioceptive demands in learning and maturation of jaw motor skills. Similarly, well-developed muscle spindles in young biceps reﬂect early need of reﬂex control in learning and C 2011 performing arm motor behavior. Anat Rec, 294:683–693, 2011. V Wiley-Liss, Inc. Key words: ﬁber types; intrafusal ﬁbers; jaw muscle; skeletal muscle Grant sponsors: Department of Odontology (Medical Faculty of Umeå University), Västerbotten County Council, Swedish Dental Society. *Correspondence to: Per-Olof Eriksson, Department of Odontology, Clinical Oral Physiology, Faculty of Medicine, Umeå University, Umeå S-901 87, Sweden. Fax: (þ46) – 90 13 25 78. E-mail: email@example.com C 2011 WILEY-LISS, INC. V Received 29 June 2010; Accepted 10 December 2010 DOI 10.1002/ar.21347 Published online 2 March 2011 in Wiley Online Library (wileyonlinelibrary.com). 684 ÖSTERLUND ET AL. INTRODUCTION Control of jaw muscle function relies on selective recruitment of motor units [cf. (Stålberg et al., 1986; Hannam and McMillan, 1994)] and sensory information from muscle, joint, periodontal, skin and mucosal mechanoreceptors (Bradley, 1995). Of muscle receptors, muscle spindles have a unique inﬂuence on muscle activity allowing the brain to simultaneously receive sensory message and control muscle spindle length and change in length (Rufﬁni, 1898; Boyd, 1962; Banks, 1994; Zelená, 1994). Although far outnumbered by extrafusal ﬁbers, muscle receptors are equal to extrafusal ﬁbers in amount of nervous trafﬁc (Matthews, 1981b). Muscle spindles play multifunctional roles contributing in proprioception, postural and movement control, motor learning and in plasticity of motor behaviors (Windhorst, 2007, 2008), in predicting future kinematic states by acting as forward sensory model (Dimitriou and Edin, 2010), and as suggested, in the genesis and spread of chronic muscle pain (Johansson et al., 1999; Blair et al., 2003; Johansson et al., 2003). They consist of a bundle of intrafusal ﬁbers enclosed in a fusiform connective tissue capsule attached in parallel with the extrafusal ﬁbers. The intrafusal ﬁbers have been classiﬁed based on their enzyme-histochemical staining pattern (Ovalle and Smith, 1972), their location of nuclei and their physiological properties (Boyd, 1962; Matthews, 1981a; Boyd and Smith, 1984; Barker and Banks, 1994) into dynamic nuclear bag (bag1), static nuclear bag (bag2) and static nuclear chain (chain) ﬁbers. Furthermore, a fourth type acid stable bag1 (AS-bag1) is described in the adult masseter (Eriksson and Thornell, 1985, 1990; Eriksson et al., 1994). It is well established that adult human jaw muscles differ from limb muscles in composition and distribution of muscle ﬁber types and MyHC isoforms (Eriksson, 1982; Eriksson and Thornell, 1983; Sciote et al., 1994; Stål et al., 1994; Monemi et al., 1999; Korfage et al., 2005a,b). It has also been shown that human jaw muscles in adults diverge from limb muscles in number and morphology of muscle spindles. Thus, the adult human masseter muscle contains especially large and complexly arranged muscle spindles (Freimann, 1954; Voss, 1971; Eriksson and Thornell, 1985, 1987, 1990; Eriksson et al., 1994; Eriksson et al., 1995). Typical features are a high muscle spindle density, large capsule diameter, a high number of intrafusal ﬁbers per spindle and numerous compound spindles, that is, clusters of spindles located closely together within a common capsule. In line with regional differences in the extrafusal ﬁber type composition, the muscle spindles are also heterogeneously distributed. The largest and most complex spindles are located in the deep masseter, which is composed mainly of type I (Eriksson and Thornell, 1983) ﬁbers or slow twitch motor units (Stålberg and Eriksson, 1987). A recent study showed that the human masseter is regionally differentiated in ﬁber type composition already at 3 years of age (Österlund et al., in press). The study also revealed signiﬁcant changes in the extrafusal ﬁber population from young age to adulthood. Since motor and sensory control systems of muscle are interlinked, we hypothesize that also muscle spindles undergo changes in composition and distribution from young age to adulthood. To address this question, the present study examined muscle spindles in the young masseter muscle, and compared the results with previously reported data on the adult masseter (Eriksson and Thornell, 1985, 1987, 1990; Eriksson et al., 1994; Eriksson et al., 1995). For comparison muscle spindles in the young biceps brachii were included in the study, and likewise compared with adult biceps muscle spindles (Liu et al., 2002). MATERIALS AND METHODS Muscle Specimens Muscle samples were collected at autopsy 1–3 days post mortem, a delay that does not hamper reliable ﬁber typing (Eriksson et al., 1980). Two specimens from each of the anterior (sup ant) and posterior (sup post) superﬁcial portions, and the deep (deep) portion of the human masseter muscle, were obtained from seven previously healthy young subjects with normal bite and jaw-face morphology, four males aged 3, 4, 4, and 6 years, and three females aged 3, 7, and 7 years. In addition, one specimen was obtained from the biceps brachii muscle (biceps) of the same subjects. There were missing samples for the sup ant (male 4y), and biceps (female 7y). The samples were collected before 1990 according to prevailing directions issued by the National Board of Health and Welfare Stockholm, Sweden. The ﬁndings were compared with previously reported data from muscle spindles in the adult masseter (Eriksson and Thornell, 1987, 1990; Eriksson et al., 1995) and adult biceps brachii muscles (Liu et al., 2002). Enzyme-Histochemistry The muscle samples were rapidly frozen in propane chilled with liquid nitrogen and stored at 80 C. Serial 10 lm cross-sections were cut at –20 C in a cryostat microtome and treated with an antibody against slow tonic myosin heavy chain (ALD 19) (Sawchak et al., 1985) for detection of muscle spindles/intrafusal ﬁbers, and processed for myoﬁbrillar adenosine triphosphate (mATP) at pH 10.3, 4.6, and 4.3 for classiﬁcation of intrafusal ﬁbers. Intrafusal ﬁbers were classiﬁed into four types of ﬁber bag1, acid stable bag1 (AS-bag1), bag2 and chain ﬁbers on the basis of their mATPase staining intensity. Fibers with no or weak staining at pH 10.3, 4.6, and 4.3 (alkaline- and acid-labile reaction) were termed bag1 ﬁbers. Fibers with no or weak staining at pH 10.3 (alkaline-labile reaction) to strong staining at pH 4.6 and 4.3 (acid stable reaction) were termed acid stable bag1 (AS-bag1). Fibers with moderate to strong staining intensity at pH 10.3, 4.6, and 4.3 (alkaline- and acid stable reaction) were termed bag2 ﬁbers, and ﬁbers with strong staining at pH 10.3 (alkaline stable reaction) and 4.6 (acid stable reaction) and no or weak staining at pH 4.3 (acid-labile) were termed chain ﬁbers (Eriksson and Thornell, 1990). This classiﬁcation of intrafusal ﬁber types was performed in samples of three masseter muscles (female 3 years, male 4 years and female 7 years) and all six biceps muscles. 685 MASSETER MUSCLE SPINDLES AT YOUNG AGE TABLE 1. Muscle spindle (MS) density, number of single and compound muscle spindles, total number of intrafusal ﬁbers (IF) and number of intrafusal ﬁbers per muscle spindle detected in seven masseter and six biceps brachii muscles Muscle spindles (MS): Total number Density MS/cm2 (SD) Number in A, AB and B regions Number in C-region Single MS: Number Proportion (%) Compound MS: Number Proportion (%) Intrafusal ﬁbres (IF): Total number IF per single MS: Mean (SD) Median Min-max IF per compound MS: Mean (SD) Median Min-max Sup ant Sup post Deep Masseter Biceps 224 27 (11) 89 101 22 4 (6) 8 12 378 43 (16) 144 190 624 25 (8) 241 303 97 20 (13) 54 34 80 90 8 100 110 76 198 82 52 96 9 10 0 0 34 24 43 18 2 4 800 46 1609 2455 434 8.0 (3) 7 1–23 6.4 (2) 6 3–10 9.4 (2) 9 2–21 8.3 (4) 8 1–23 7.4 (2) 7 3–19 18.8 (9) 17 7–38 22 (1) 22 22–23 Morphometric Analyses Serial cross sections from enzyme-histochemistry were analyzed using a microscope (Leica DMR) connected with a digital camera (Leica DC 220). The morphometric analyses, including muscle spindle density, compound muscle spindle structure and number of intrafusal ﬁbers per muscle spindle was performed in samples of all masseter (n ¼ 7) and biceps (n ¼ 6) muscles. Muscle spindle density was measured as number of muscle spindles per area (MS/cm2). Deﬁnition of compound muscle spindle structure is when two or more spindles share the same capsule. The muscle spindle can be divided into three morphological regions. The capsular regions the A region including the equator and the juxta-equatorial parts, containing the paraxial ﬂuid space; the B region extending from the end of the paraxial ﬂuid space to the end of the capsule; and the extra capsular C region (Banks, 1994; Soukup et al., 2003). We also deﬁne a transitional AB region in between the A and B regions. Number of intrafusal ﬁbers per muscle spindle and capsule diameter were measured in muscle spindle regions A, AB and B, from three masseter muscles and from all biceps muscles (n ¼ 6). The muscle spindle capsule and intrafusal ﬁber diameters were measured on the computer screen processed in image analyses system (Leica QWin). Incomplete serial sections were excluded from analysis. Statistical Analysis Group data were presented as means and standard deviations (SD). Paired t test and unpaired Mann-Whitney U test were used to test the null hypothesis (H0) of no differences in density, muscle spindle capsule diameter, intrafusal ﬁbers per muscle spindle, proportion of intrafusal ﬁber types, and intrafusal ﬁber diameter between masseter portions, masseter and biceps brachii muscles and between young and adult masseter muscles. H0 was rejected at the level of signiﬁcance P 0.05. RESULTS In total 624 muscle spindles were detected in the masseter muscle. Of these, 34 in the sup ant, two in the sup post and 44 in the deep masseter were excluded for further analysis due to incomplete serial sections. Of the remaining 544 spindles, 241 were sectioned in the capsular regions A, AB and B, and 303 in the C-region. Of the 241 spindles sectioned in the capsular regions, 198 were deﬁned as single and 43 as compound spindles, altogether containing 2455 intrafusal ﬁbers (1649 in single and 806 in compound spindles). In the biceps, a total of 97 muscle spindles were detected, of which nine were excluded due to incomplete sections. Of the remaining 88 spindles, 54 were sectioned in the capsular regions and 34 in the C-region. Of the 54 biceps spindles, 52 were deﬁned as single and two as compound spindles, altogether containing 434 intrafusal ﬁbers (389 in single and 45 in compound spindles) (Table 1). Muscle Spindle Density The muscle spindle density, MS/cm2, was higher (mean, SD) in the deep 43 (16) than in the sup ant 27 (11) and sup post 4 (6) masseter portions (P ¼ 0.045 and 0.001, respectively), and higher in the sup ant than in the sup post (P ¼ 0.002). The muscle spindle density in the biceps brachii 20 (13) was lower than that of the deep (P ¼ 0.044) masseter (Table 1). Compound Muscle Spindles Compound muscle spindles (Fig. 1) were seen in the deep, 24%, and sup ant, 10%, masseter portions, and the biceps brachii, 4% (Table 1). 686 ÖSTERLUND ET AL. Fig. 1. Cross-sections of two masseter compound muscle spindles (subjects aged 3 and 4 years) stained for mATPase at pH 10.3. Spindle from the superﬁcial (a) and deep (b) portions with 30 and 37 intrafusal ﬁbers, respectively. Bar ¼ 100 lm. Number of Intrafusal Fibers per Muscle Spindle Table 1 summarizes the number of intrafusal ﬁbers (mean, median and min-max) of single and compound muscle spindles in the masseter and biceps. The mean number of intrafusal ﬁbers of single muscle spindles was signiﬁcantly larger in the deep masseter, 9.4, than in the biceps, 7.4 (P ¼ 0.03). Figure 2 shows the different distributions of the relative frequency of muscle spindles (%) versus number of intrafusal ﬁbers for single and compound spindles, respectively. Muscle Spindle Capsule Diameter Measurement of capsule diameter in the masseter was based on three muscles containing 126 muscle spindles (104 single and 22 compound spindles) sectioned in the A, AB and B capsular regions. Out of these 86 single and 22 compound spindles were measured. Out of 52 single and two compound spindles in the biceps, 36 single and two compound were measured. In the masseter, the mean capsule diameter of single muscle spindles (lm, mean SD) in regions A, AB and B were, 162 (55), 112 (29), and 72 (16), respectively. The corresponding values for the biceps were, 118 (25), 93 (14), and 56 (15), respectively. For both the masseter and biceps, the diameters of muscle spindle regions A and AB were signiﬁcantly larger than that of region B (masseter P ¼ 0.010 and P ¼ 0.024, biceps P ¼ 0.041 and P ¼ 0.013, respectively). The diameter of region A was signiﬁcantly larger than that of region AB (masseter P ¼ 0.035, biceps P ¼ 0.035). In the masseter, the mean capsule diameter (lm, mean SD) of pooled data for single muscle spindles (n ¼ 86) was 96 (37) and in the biceps (n ¼ 36), 76 (28). There were no signiﬁcant differences in capsule diameter between masseter portions or between masseter and biceps. The mean capsule diameter of compound muscle spindles (lm, mean SD) was 168 (60) in the masseter (n ¼ 22) and 134 (10) in the biceps (n ¼ 2). Intrafusal Fiber Types In the masseter, classiﬁcation of intrafusal ﬁber types was based on 126 muscle spindles sectioned in the A, AB, B and C regions (32 in sup ant, six in sup post and 88 in deep masseter), containing 1251 intrafusal ﬁbers (246 in sup ant, 29 in sup post and 976 in the deep masseter). Of these, 1091 (240 in sup ant, 29 in sup post and 822 in deep masseter) were analysed in all serial sections. In the biceps, 422 out of 434 intrafusal ﬁbers were classiﬁed. Bag1, AS-bag1, bag2 and chain ﬁbers were identiﬁed in both muscles (Fig. 3). A variable pattern of ATPase staining was seen at all pH levels and in all muscle spindle regions (Fig. 4). In both muscles, for all pH levels, the bag1 ﬁbers were unstained or weakly to moderately stained in the A and B regions and moderately stained or unstained in the C region. AS-bag1 ﬁbers were weakly to moderately stained or unstained at pH 10.3 and moderately to strongly stained at pH 4.6 and 4.3. Bag2 ﬁbers were in all regions strongly to moderately stained at pH 10.3 and 4.6 and moderately to weakly stained at pH 4.3. Chain ﬁbers were strongly or moderately stained at pH 10.3 and 4.6 and unstained at pH 4.3. In both muscles, the bag1 ﬁber staining was stronger in the Cregion versus other regions. No regional differences were detected in bag2 and chain ﬁbers. Intrafusal Fiber Type Population Of all masseter spindles (n ¼ 126), 90% contained bag1, 15% AS-bag1, 76% bag2, and 96% chain ﬁbers. The corresponding values for the biceps brachii were 96%, 45%, 28%, and 100%. In the masseter, 65% of the spindles contained the three-ﬁber types bag1, bag2 and chain ﬁbers and 10% contained all four ﬁber types. The corresponding values for the biceps were 28% and 4%. Compared with the biceps, the masseter contained signiﬁcantly more bag2 ﬁbers, 16% versus 4% (P ¼ 0.008) and less chain ﬁbers, 52% versus 64% (P ¼ 0.024) (Fig. 5). The average composition of intrafusal ﬁber MASSETER MUSCLE SPINDLES AT YOUNG AGE 687 Fig. 2. Distribution of relative frequency of muscle spindles (%) versus number of intrafusal ﬁbers (IF) in single and compound masseter spindles and single biceps spindles. Note for the masseter, difference in distribution between single and compound spindles indicating separate populations of muscle spindles. types in masseter single muscle spindles was 2 bag1, 1 bag2 and 4 chain, and in the biceps, 2 bag1 and 4 chain ﬁbers (Table 2). Combinations of Intrafusal Fiber Types For the masseter single muscle spindles, there were 67 different combinations of bag1, AS-bag1, bag2 and chain ﬁbers. Of these, 47 (70%) appeared once, nine twice, nine three times, one four times and one eight times. The corresponding values for the biceps were 39 combinations, of which 31 (79%) appeared once, ﬁve twice, two three times and one four times (Table 3). Data for compound masseter and biceps muscle spindles are shown in Table 3. Of the 67 different combinations in the masseter, two overlapped the sup ant and sup post masseter portions, six overlapped the sup ant and deep, three sup post and deep, and only one combination (2 bag1 and 2 chain ﬁbers) overlapped all three portions. Of the 67 masseter and 39 biceps ﬁber type combinations, thirteen were found in both muscles. A comparison between subjects showed that of the 67 masseter combi- nations, 31 (46%) were seen in subject one, 17 (25%) in subject two and 36 (53%) in subject three. Only 2 (3%) overlapped all three subjects. Of the 39 combinations in the biceps, 16 (41%) were seen in subject one, 6 (15%) in subject two, 5 (13%) in subject three, 7 (19%) in subject four, 5 (13%) in subject ﬁve and 5 (13%) in subject six, without any overlap among the six subjects. Diameter of Intrafusal Fibers The diameter of intrafusal ﬁber types in the masseter was measured in 884 out of 1223 ﬁbers (255 bag1, 22 AS-bag1, 162 bag2 and 445 chain), and for the biceps in 349 out of 434 ﬁbers (89 bag1, 25 AS-bag1, 13 bag2 and 222 chain). The diameters of the masseter intrafusal ﬁber types (lm, mean SD) were bag2 18.4 (1.6) > bag1 15.2 (1.8) > AS-bag1 14.9 (2.2) > chain 10.2 (1.2). In the biceps the diameters were bag2 17.8 (3.1) > AS-bag1 15.0 (2.4) > bag1 13.9 (2.1) > chain 8.2 (0.3). In the masseter, bag2 ﬁbers were signiﬁcantly larger than both bag1 and chain ﬁbers (P ¼ 0.028 and P ¼ 0.013, respectively). There were no differences between the masseter muscle 688 ÖSTERLUND ET AL. Fig. 3. Serial cross-sections of single spindles of the deep masseter (left) and biceps (right), stained for mATPase pH 10.3, (a, d), pH 4.6 (b, e) and pH 4.3 and (c, f). The deep masseter spindle (subject 4 years) contains 20 intrafusal ﬁbers (5 bag1, 6 bag2 and 9 chain ﬁbers þ 4 ﬁbers within the capsule tissue), and the biceps spindle (subject 3 years) nine intrafusal ﬁbers (1 bag1, 1 AS-bag1 and 7 chain ﬁbers). Fiber types bag1, AS-bag1 bag2 and chain have been labelled. Bar ¼ 50 lm. portions. Also in the biceps, bag1, AS-bag1 and bag2, were signiﬁcantly larger than chain ﬁbers (P ¼ 0.001, P ¼ 0.002 and P ¼ 0.002, respectively). Masseter bag1 ﬁbers were signiﬁcantly larger than those of the biceps (P ¼ 0.049). The mean intrafusal ﬁber diameter for pooled data of bag1, AS-bag1, bag2 and chain ﬁbers (lm, mean SD) was signiﬁcantly larger in the masseter (n ¼ 884), 13.5 (2), than in the biceps (n ¼ 349), 10.0 (1) (P ¼ 0.045). Comparison Between Young and Adult Masseter Muscle Spindles The muscle spindle density was signiﬁcantly higher (P ¼ 0.005) in young masseter, 25, than in adult (Eriksson and Thornell, 1987) masseter, 7. In both young and adult masseter, the deep portion contains the majority of spindles (young 61% and adult 74%), and compound muscle spindles occur in a signiﬁcant amount. Young 689 MASSETER MUSCLE SPINDLES AT YOUNG AGE Fig. 4. Myoﬁbrillar ATPase-staining pattern at pH 10.3, 4.6 and 4.3 of bag1, AS-bag1, bag2 and chain ﬁbers in the A, AB, B and C regions of masseter and biceps muscle spindles. Proportion (%) of unstained, and weakly, moderately or strongly stained ﬁbers. masseter did not differ from the adult (Eriksson and Thornell, 1990) in number of intrafusal ﬁbers of single spindles (young 8.3, adult 7.3), capsule diameter of single spindles (young 96 lm, adult 98 lm) or intrafusal ﬁber diameter (young 13.5 lm, adult 16 lm). The ratio between the mean bag ﬁber diameter (pooled data for bag1, AS-bag1 and bag2 ﬁbers, (n ¼ 439) and the mean diameter of extrafusal type I ﬁbers (Österlund et al., in press) was 0.8 (16.5 lm/21.7 lm) for the young masseter versus 0.4 (18.1 lm/43.9 lm) for the adult (Eriksson and Thornell, 1990). Comparison Between Young and Adult Biceps Muscle Spindles Young and adult (Liu et al., 2002) biceps muscle spindles were similar in enzyme-histochemical staining pattern of intrafusal ﬁber types, mean number of intrafusal ﬁbers per muscle spindle, heterogeneity in combinations of intrafusal ﬁber types and lack of bag2 ﬁbers. DISCUSSION Fig. 5. Relative proportion (%) of intrafusal ﬁber types bag1, ASbag1 bag2 and chain in the spindle populations of young masseter and biceps. Data based on average values for three masseter and six biceps muscles. Note, more bag2 and less chain ﬁbers in the masseter. The main ﬁnding of this study was the general resemblance in muscle spindle morphology between young masseter and adult masseter (Eriksson and Thornell, 1987, 1990; Eriksson et al., 1995). Hence, contrary to 690 ÖSTERLUND ET AL. TABLE 2. Average composition (mean, median, and min-max) of intrafusal ﬁber types in single and compound masseter and biceps muscle spindles (MS) Single MS Masseter Mean (SD) Median Min-max Biceps Mean (SD) Median Min-max Compound MS Bag1 AS-Bag1 Bag2 Chain Bag1 AS-Bag1 Bag2 Chain 2.0 (2) 2 0–8 0.2 (0) 0 0–3 1.0 (1) 1 0–5 4.0 (4) 4 0–12 5.5 (3) 6 0–11 0.2 (1) 0 0–4 3.4 (3) 2 3–12 7.7 (5) 7 5–20 1.8 (1) 2 0–5 0.5 (1) 0 0–4 0.3 (1) 0 0–2 4.8 (2) 4 1–14 7.0 (1) 7 6–8 1.5 (1) 2 1–2 0.5 (1) 1 0–1 13.5 (4) 14 12–15 our hypothesis, masseter muscle spindles do not undergo major changes in morphology and composition and distribution of intrafusal ﬁber types from young age to adulthood. The result was similar for the biceps muscle, that is, young spindles were alike adult spindles (Liu et al., 2002). These ﬁndings show that the human masseter and biceps muscle spindles are differentiated and morphologically mature already at young age. The current result contrasts our recent ﬁndings of signiﬁcant differences between young and adult masseter extrafusal ﬁber type compositions (Österlund et al., in press). We therefore conclude that muscle spindles precede the extrafusal ﬁber population in growth and maturation. This in turn suggests early proprioceptive demands during growth and maturation of jaw motor skills. Correspondingly, well-developed muscle spindles in young biceps brachii reﬂect signiﬁcant need of reﬂex control in learning and performing arm motor behavior. Nevertheless, the more complex morphology of young masseter spindles versus young biceps spindles indicates a more advanced proprioceptive control of the masseter muscle. Notably, difference between masseter single and compound spindles in distribution of frequency of spindles versus number of intrafusal ﬁbers indicates two separate populations of muscle spindles in young masseter. Young and adult masseter muscle spindles were similar in containing exceptionally large capsule diameter and high number of intrafusal ﬁbers, high frequency of compound spindles and heterogeneous distribution and most complexly arranged spindles located in the deep masseter. However, the muscle spindle density was three times higher in the young than in the adult (Eriksson and Thornell, 1987) masseter. Muscle spindle density varies signiﬁcantly between different muscles (Boyd and Smith, 1984) and can be regarded as an indicator of functional differences. Muscles active in gross movements have low spindle density whereas muscles initiating ﬁne movements or maintaining postural stability have a high spindle density, for example, the extra ocular (Bruenech and Ruskell, 2001; Boyd-Clark et al., 2002), lumbrical (Soukup et al., 2003) and neck (BoydClark et al., 2002; Liu et al., 2003) muscles. Given that the extrafusal ﬁber diameter is about two times larger in the adult than in the young masseter (Österlund et al., in press) the adult masseter volume is about twice that of the young masseter. Therefore, since young and adult spindles are of similar size, an age-related decrease in spindle density is explainable. An additional marker for comparing young and adult spindles is the ratio between intrafusal and extrafusal ﬁber diameter. This ratio was larger in the young than in adult masseter, 0.8 versus 0.4. Likewise, for the biceps, corresponding ratios were 0.6 and 0.3. Taken together, these data suggest larger functional inﬂuence of muscle spindles at young age, especially for the masseter muscle. Both the masseter and biceps were strikingly heterogeneous in allotment of types and number of intrafusal ﬁbers, and the ‘‘average’’ composition of intrafusal ﬁber types (Table 2) should not be taken as ‘‘the typical’’ muscle spindle appearance. Rather, most spindles were unique in the combination of intrafusal ﬁber types, that is, the majority of combinations occurring only once. Furthermore, only one intrafusal ﬁber type combination overlapped all three masseter portions, and few combinations overlapped the young masseter and biceps. Also individuals were unique in ﬁber type composition of muscle spindles, with hardly any overlapping combinations between subjects. Although noted in a limited number of subjects, this variability in morphology between spindles, muscle portions, muscles and subjects suggest highly differentiated proprioceptive abilities allocated in governing mandibular and arm positions and movements. The present data showed that lack of bag2 ﬁbers was common in the young masseter and biceps muscle spindles. Spindles lacking bag2 ﬁbers have previously been reported for the human adult biceps brachii (Liu et al., 2002) and deep neck (Liu et al., 2003) muscles. Bag1 ﬁbers are known to play a major role in the production of the velocity-sensitive (dynamic) response in the primary endings whereas bag2 ﬁbers mediate the length (static) sensitivity along with chain ﬁbers (Boyd, 1980; Matthews, 1981a; Barker and Banks, 1994). Furthermore, ﬁve of the young masseter spindles (4%) lacked chain ﬁbers, but of these, all contained bag1 and four contained bag2 ﬁbers, suggesting ability for both dynamic and static sensitivity. Taken together, lack of bag2 and chain ﬁbers would again reﬂect diversity in physiological qualities between spindles and muscles, preferentially in static length sensitivity properties. AS-bag1 ﬁbers, described previously in the adult masseter (Eriksson and Thornell, 1985, 1987, 1990; Eriksson et al., 1994) but not in the biceps, were more frequent in the young biceps. Its physiological characteristics and role in proprioceptive control remains to be examined. Reﬂex mediated stiffness contributes to the maintenance of mandibular posture relative to the maxilla during locomotion (Miles et al., 2004; Miles, 2007). It has been hypothesized that the jaw stretch reﬂex may play an important role in contributing to the stiffness of the 691 MASSETER MUSCLE SPINDLES AT YOUNG AGE TABLE 3. Combinations of intrafusal ﬁber types in single and compound masseter and biceps brachii muscle spindles. In the masseter single muscle spindles, there were 67 combinations of intrafusal ﬁber types, in the biceps 39. In the masseter compound muscle spindles, there were 22 combinations of intrafusal ﬁber types, in the biceps two Masseter single MS Combinations 1 1 1 1 2 1 1 1 1 2 1 1 1 5 5 3 1 1 1 1 6 3 1 3 2 3 5 5 1 1 4 P Bag1 AS-Bag2 Bag2 Chain 8 7 6 5 5 4 4 4 4 4 4 3 3 3 3 3 2 2 2 2 2 2 1 1 1 1 1 1 0 0 0 0 l 0 1 0 3 1 0 0 0 0 2 0 0 0 0 2 1 1 1 0 0 1 1 1 0 0 0 2 1 0 2 3 0 1 1 1 0 3 2 1 0 1 3 2 1 0 1 5 3 1 1 0 3 1 0 2 1 0 1 1 1 12 7 9 11 3, 12 4 9 7 0, 7 3, 4 5 2 3 1, 2, 3, 5, 6 2, 4, 5, 6, 8 3, 5, 8 3 8 6 5 1, 3, 4, 5, 6, 11 2, 3, 4 4 1, 2, 3 3, 4 0, 4, 8 2, 4, 5, 7, 10 0, 1, 3, 4, 5 3 3 0, 2, 3, 4 67 Biceps single MS Combinations 2 1 1 2 2 3 1 4 3 3 1 6 4 4 2 P Bag1 AS-Bag1 Bag2 Chain 5 4 3 3 3 3 2 2 2 2 1 1 1 1 0 0 1 2 0 0 0 4 1 0 0 1 1 0 0 1 0 0 0 2 1 0 0 0 1 0 1 0 1 0 0 6, 7 5 7 2, 14 5, 8 5, 6, 7 5 3, 4, 5, 6 5, 7, 8 2, 4, 5 3 1, 3, 4, 5, 6, 7 3, 4, 8, 9 2, 3, 4, 6 2, 3 39 mandible in speech production (Smith, 1992). During fetal life survival reﬂexes are gradually succeeded by automatic postural reﬂexes in neonatal life. The trigeminal sensori-motor system develops early in neonatal period (Jääskälainen, 1993). Actually, the earliest reﬂex found Table 3. (continued) Masseter compound MS Combinations 1 1 1 1 1 1 1 1 2 1 1 2 1 1 1 1 1 1 1 P Bag1 AS-Bag1 Bag2 Chain 11 11 9 9 7 7 7 7 6 5 5 4 4 3 3 2 1 1 0 0 0 1 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 7 2 10 5 0 1 5 2 2 2 1 2 3 6 3 1 4 1 12 20 14 8 12 10 5 8 3 1, 6 4 4 6, 13 7 1 10 5 2 10 14 22 Biceps compound MS Combinations 1 1 P Bag1 AS-Bag1 Bag2 Chain 8 6 1 2 1 0 12 15 2 in the human embryo is the trigemino-neck reﬂex. A head withdrawal effect from stimulation of the upper lip has been detected already at the seventh gestational week (Humphrey, 1952). Studies in neonates, infants and children have shown that reﬂex response properties change dramatically with development. For both myotatic and cutaneous reﬂexes, ‘‘mature’’ patterns do not emerge until late childhood (Finan and Smith, 2005). These functional changes occur in parallel with substantial morphological transformations of the skeleton, muscles and nervous system. The morphological and functional changes may also include modiﬁcation of the functional role of reﬂexes coincident with developing motor skills. Our present ﬁnding of a general resemblance between young and adult masseter muscle spindles suggests that the morphological basis for reﬂex jaw motor control in learning and improving jaw motor tasks is well differentiated and matured already at young age. In both the adult (Eriksson and Thornell, 1987) and young masseter, the deep portion contained the highest spindle density and the most complex spindles. This portion is composed of vertically running ﬁbers and a preponderance of type I ﬁbers, which build up slow twitch, low threshold motor units. Therefore, its extrafusal and intrafusal ﬁbers in combination make the deep portion well adapted for postural mandibular control, possibly of special importance in early learning and improving speech function. In speech, precise jaw movements occur within a three-dimensional space around the mandibular postural position. Interestingly, the number of muscle spindles in the jaw muscles seems to increase in the evolutionary series from lower primates towards man (Kubota and Masegi, 1975, 1977). This observation 692 ÖSTERLUND ET AL. suggests changes during evolution towards stronger proprioceptive impact on jaw motor control as for movements in mastication and learning and production of speech, a function evolved only in man. Previous ﬁndings of coordinated mandibular and head-neck movements during natural jaw-opening closing tasks suggest a functional interlinkage between the jaw and the neck sensori-motor systems. Studies also indicate that this functional connection between the jaw and neck sensori-motor systems is innate (c.f. Zafar et al., 2000; Häggman-Henrikson and Eriksson, 2004; Eriksson et al., 2007). Furthermore, there is experimental evidence of reﬂex connections between the masseter and neck muscles (Hellström et al., 2000). To evaluate the functional maturation of the jaw-neck motor coupling at young age we have examined concomitant mandibular and head-neck movements during jaw openingclosing motor activities in 5–6-year-old children. The results contrast ﬁndings in adults. Adults show well coordinated and regular spatiotemporal patterns of both mandibular and head-neck movements, whereas movement patterns in children are irregular and variable (Zafar et al., unpublished results). 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