Sage Journals: Discover world-class research

Abstract

Muscle spindle density is extremely high in the deep muscles of the human neck. However, there is a paucity of information regarding the morphology and immunoreactivity of these muscle spindles. The objective of this study was to investigate the intrafusal fiber content and to assess the myosin heavy chain (MyHC) composition of muscle spindles from human deep neck muscles. In addition to the conventional spindles containing bag₁, bag₂, and chain fibers (b₁b₂c spindle), we observed a number of spindles lacking bag₁ (b₂c spindle) or bag₂ (b₁c spindle) fibers. Both bag₁ and bag₂ fibers contained slow tonic MyHCs along their entire fiber length and MyHCI, MyHCIIa, embryonic, and α-cardiac MyHC isoforms along a variable length of the fibers. Fetal MyHC was present in bag₂ fibers but not in bag₁ fibers. Nuclear chain fibers contained MyHCIIa, embryonic, and fetal isoforms with regional variations. We also compared the present data with our previous results obtained from muscle spindles in human biceps brachii and the first lumbrical muscles. The allotment of numbers of intrafusal fibers and the MyHC composition showed some muscle-related differences, suggesting functional specialization in the control of movement among different human muscles.

Keywords

human muscle spindle deep neck muscles mATPase MyHC intrafusal fibers

Muscle spindles are important for the control of movement and posture in mammals. Mice lacking these receptors are unable to support their weight and have an abnormal posture (Ernfors et al. 1994; Walro and Kucera 1999). Muscle spindles are found in practically all mammalian skeletal muscles, and they sense muscle length and changes in muscle length (Barker and Banks 1994). Muscle spindles have been proposed to play a major role in increasing muscle tone and in the pathophysiology of muscle pain syndromes (Johansson and Sojka 1991; Djupsjobacka et al. 1995; Matre et al. 1998; Johansson et al. 1999). Some muscles with a high spindle density are involved in chronic muscle pain syndromes (Johansson and Sojka 1991; McPartland et al. 1997).

Muscle spindles contain specialized fibers, called intrafusal fibers, innervated by both sensory neurons and γ-motor neurons. There are three types of intrafusal fibers, nuclear bag₁, nuclear bag₂, and nuclear chain fibers, classified according to their distinct myosin ATPase (mATPase) reactions (Ovalle and Smith 1972). The morphological features of muscle spindles in different human muscles are more diverse than in other mammals. We have reported that the human muscle spindles in different muscles exhibit distinct features that most likely reflect the functional demands of these particular muscles (Eriksson and Thornell 1987,1990; Eriksson et al. 1994; Liu et al. 2002; Soukup et al. 2003). In human biceps brachii muscle, the muscle spindle density is much lower than in jaw and lumbrical muscles but each biceps spindle has a unique morphological identity (Liu et al. 2002). In contrast, spindles in the human masseter are exceptionally large and are more complex than those of limb muscles (Eriksson and Thornell 1987,1990). The deep muscles of the human neck, i.e., the rectus capitis posterior major, the rectus capitis posterior minor, the obliquus capitis inferior, and the obliquus capitis superior, are located deep in the suboccipital region. These muscles are quite small and short. They function in fine rotatory movements of the head and help maintain the stability of the cervical spine. The deep neck muscles have an unusually high muscle spindle density (Cooper and Daniel 1963; Peck et al. 1984; Kulkarni et al. 2001), almost five times higher than that of the splenius capitis and three times that of the semispinalis capitis muscle (Peck et al. 1984). However, the intrafusal fiber content and MyHC composition of muscle spindles from the deep muscles of the neck have not been studied in detail. The MyHC composition is considered the best marker for the functional properties of human skeletal muscle fibers because it correlates with contraction force and velocity (Larsson and Moss 1993; Bottinelli and Reggiani 2000; Bottinelli 2001).

In an attempt to correlate muscle spindle structure and function in human muscles, the fiber type content, general morphology, and MyHC composition of muscle spindles in the deep muscles of the human neck were examined and compared with the data available for other human muscles.

Materials and Methods

Deep muscles of the neck (rectus capitis posterior major, rectus capitis posterior minor, obliquus capitis inferior and obliquus capitis superior) were collected at autopsy from two females, ages 26 and 17, three males, ages 55, 21, and unknown, without known muscle related disorders. The samples were collected according to the ethical recommendations of the Swedish Transplantation Law, with the approval of the Medical Ethical Committee, Umeå University.

The muscle samples were mounted, rapidly frozen in propane chilled with liquid nitrogen, and stored at −81C until sectioned. Frozen specimens were serially sectioned at −23C using a Reichert Jung cryostat (Leica; Nussloch, Germany). Sections to be used for demonstration of mATPase activity after preincubation at pH 10.4, 4.6, and 4.3 (Dubowitz 1985) were 8 μm thick; those for immunocytochemistry were 5 μm thick.

Antibodies and Labeling

Monoclonal antibodies specific for different MyHC isoforms were used (Table 1). The specificities of these antibodies have been carefully assessed for human muscles (Liu et al. 2002). The short names of antibodies are listed in Table 1. All antibodies were diluted in 0.01 M PBS containing 0.1% bovine serum albumin (BSA) and were used at their optimal dilution.

Immunocytochemistry

Sections were processed for immunocytochemistry as previously described (Liu et al. 2002), using the indirect peroxidase-anti-peroxidase (PAP) technique (Sternberger 1979). The PAP products were purchased from Dakopatts (Glostrup, Denmark). Incubation with the appropriate primary antibody was performed for 1 hr at 37C. In control sections, the primary antibody was replaced by normal serum from the same species and at the same dilutions. No staining was observed in these sections.

All sections were examined and photographed with a Nikon microscope (Eclipse, E800; Tokyo, Japan) equipped with a Spot camera (RT color; Diagnostic Instruments, Sterling Heights, MI).

Survey

Twenty-two muscle specimens were sectioned and stained. A total of 199 muscle spindles were examined, and 36 of these were studied in consecutive serial transverse sections covering a distance of approximately 2 mm. Three regions were examined: the A region, including equator and juxtaequatorial parts, containing the periaxial fluid space; the B region, extending from the end of the periaxial fluid space to the end of the capsule; and the C region, corresponding to the extracapsular portion of the spindle (Barker and Banks 1994). Each region was further subdivided into an inner (proximal to the equator) and outer (distal to the equator) portion.

Statistical Analyses

StatView software (3rd ed; SAS Institute, Cary, NC) was used for statistical analysis. Statistic comparison of the intrafusal fiber numbers between different muscles was performed with an unpaired t-test. Differences were considered significant at p<0.05.

Results

General Morphological Features

The 199 muscle spindles studied contained a total of 1039 intrafusal fibers. Two hundred and two fibers were classified as bag₁ fibers, 185 as bag₂, and 649 as chain fibers. The remaining three fibers were considered as exceptions because of their abnormal histochemical or immunocytochemical staining patterns (see below). The number of intrafusal fibers per spindle varied considerably between four and 15 (Table 2). On average, there were 7.2 (range 4–15) fibers per spindle and in each spindle there were 1.3 (range 0–3) bag₁, 1.0 (range 0–4) bag₂, and 4.9 (range 2–11) chain fibers.

Although most spindles studied contained all three fiber types (b₁b₂c spindle), 13% of the spindles contained only one type of bag fiber (Figure 1); 10.4% lacked bag₂ fibers (b₁c spindle) whereas 2.6% lacked bag₁ (b₂c spindle). Two of 12 b₁c spindles were part of two pairs of paired spindles, whereas the remaining were single receptors. These b₁c spindles were found in three different subjects.

Only 20 of 116 muscle spindles examined in the A and/or B regions, where the full complement of intrafusal fibers is present, presented a unique allotment of numbers of bag₁, bag₂, and chain fibers. Twenty-one muscle spindles had exactly the same intrafusal fiber content: one bag₁, one bag₂, and four chain fibers.

Table 1

Monoclonal antibodies used to detect different MyHC isoforms

Antibody	Source of antigen	Specificity	Short name	Reference
ALD19 a	Chicken	MyHC slow tonic	Anti-MyHCsto	Sawchak et al. 1985
				Thornell et al. 1989
A4.840 b	Human	First developmental MyHCI	Anti-MyHCI/1st	Hughes et al. 1993
A4.951 b	Human	Second developmental MyHCI	Anti-MyHCI/2nd	Hughes et al. 1993
N2.261 b	Human	Third developmental MyHCI, MyHCIIa g	Anti-MyHCI/3rd+IIa^∗	Hughes et al. 1993
A4.74 b	Human	MyHCIIa	Anti-MyHCIIa	Hughes et al. 1993
BF35 c	Bovine	MyHCI, MyHCIIa h	Anti-MyHC“all except IIx”	Schiaffino et al. 1989
2B6 d	Rat	MyHCembryonic	Anti-MyHCemb	Gambke and Rubinstein 1984
F88 e	Human	MyHCα-cardiac	Anti-MyHCα-c	Leger et al. 1985
NCL-MHCn f	Rabbit	MyHCfetal	Anti-MyHCfet	Ecob-Prince et al. 1989

^aGift from Dr. Donald A. Fischman (Cornell University; New York, NY).

^bPurchased from Developmental Studies Hybridoma Bank (Department of Biological Sciences, University of Iowa; Iowa City, IA).

^cGift from Prof. S. Schiaffino (University of Padova; Padova, Italy).

^dGift from Prof. Neal A. Rubinstein (University of Pennsylvania School of Medicine; State College, PA).

^eGift from Dr. Jean J. Leger (Institut National de la Sante et de la Recherche Medicale, Unite 249; Montpellier, France).

^fPurchased from Novocastra Laboratories (Newcastle upon Tyne, UK).

^gOriginally reported specificity, yet it also reacts with MyHCeom and MyHCα-c in human extraocular and heart muscle, respectively (Liu et al. 2002).

^hOriginally reported to react with MyHCI, MyHCIIa, and MyHCIIb in rat muscle (Schiaffino et al. 1989). Liu et al. (2002) reported that it also reacts with human MyHCI, MyHCIIa, MyHCeom, MyHCα-c, MyHCfet, MyHCemb, and with MyHCsto in chicken anterior latissimus dorsi muscle.

In transverse sections, nuclear bag fibers were in general much larger than nuclear chain fibers. The diameter of the two types of bag fibers was most often quite similar and rather constant along their length. However, some fibers exhibited dramatic changes in diameter along their length (Figure 2). The nuclear bag fibers usually extended beyond the capsule, whereas the chain fibers usually ended within the capsule, yet some exceptions were observed. In seven spindles, bag₂ fibers terminated surprisingly in the A region at least in one pole, whereas the bag₁ and chain fibers continued further along the spindle. In one spindle, we observed nine chain fibers together with one bag₁ and one bag₂ fiber in the C region.

Most spindles appeared as single isolated receptors, but 22% of the total number of muscle spindles and 36% among those followed along serial consecutive sections occurred in different forms of linkage. The linked forms observed in the present study generally fitted the previous descriptions of parallel and paired muscle spindles in cat neck muscles (Richmond and Abrahams 1975; Bakker and Richmond 1981,1982). The latter was the most commonly found in the present study. Two of 36 spindles followed in serial consecutive sections showed a third type of complex form. Each of these two spindles was first encountered in the C region, where it contained one bag₁ and one bag₂ fiber. The bag₁ fiber tapered off and finally disappeared in the C region. The remaining bag₂ fiber continued and a new bag₁ fiber appeared, either in the C region or in the B region, of the new spindle. This new spindle, consisting of the new bag₁ fiber together with the common bag₂ fiber and five chain fibers, was then followed along the B and A regions. In addition to these three complex forms, one spindle had an extension of connective tissue from the spindle capsule which enclosed three extrafusal fibers, starting in the outer A region, for a certain distance (Figure 3).

In two spindles, at the end of the polar region, bag₁ fibers adhered to the capsular wall, exhibiting no myofibrils for a short distance, and then parted from the wall to continue further outside the capsule (Figure 4).

mATPase Activity

The mATPase staining features of nuclear bag₁, bag₂, and chain fibers at different pH values were generally in accordance with our earlier report in human biceps brachii muscle (Liu et al. 2002). Some exceptions, however, were observed. In brief, nuclear bag₁ fibers exhibited light staining along their whole fiber length at pH 10.4 (Figures 5A–5C). After preincubation at pH 4.3, these fibers showed low mATPase activity in the A region (Figure 5G) and occasionally even lost their activity in the equator. However, their mATPase activity gradually increased from low to moderate as bag₁ fibers entered the B and C regions (Figures 5H and 5I). Nuclear bag₂ fibers mostly displayed weak mATPase staining at pH 10.4 in the equatorial and juxtequatorial regions and moderate staining in the remaining parts of the fibers (Figures 5A–5C), although random variability was observed. The mATPase activity after preincubation at pH 4.3 was low in the equatorial region, low or moderate in the inner A region, and became intermediate to high along the rest of the length of the bag₂ fibers (Figures 5G–5I). In 58% of the spindles, the staining intensities of both bag₁ and bag₂ fibers at pH 4.6 were the same as at pH 4.3. In the remaining bag fibers, however, the staining intensities observed at pH 4.6 were darker than those observed at pH 4.3. Nuclear chain fibers were always darkly stained over their entire length at pH 4.6 (Figures 5D–5F) and were darkly stained in the A and inner B regions but lightly stained in the remaining parts at pH 10.4 (Figures 5A–5C). In general, chain fibers displayed low or very low mATPase activity at pH 4.3, but 28% of the chain fibers showed intermediate or high activity in the A and B regions (Figures 5G–5I).

Table 2

Intrafusal fiber type composition of individual spindles of the deep muscles of human neck a , b

N(if)	bag₁	bag₂	chain	frequency c
4	1	1	2	3 (1)
5	1	1	3	11 (2)
6	1	1	4	21 (7)
7	1	1	5	15 (3)
8	1	1	6	9 (1)
9	1	1	7	5 (2)
10	1	1	8	1
11	1	1	9	1
12	1	1	10	1
6	2	1	3	4 (1)
7	2	1	4	4
8	2	1	5	5 (1)
9	2	1	6	3 (1)
11	2	1	8	1 (1)
12	2	1	9	1 (1)
8	3	1	4	2 (1)
15	3	1	11	1
6	2	2	2	1 (1)
7	2	2	3	2 (1)
11	2	2	7	1
14	2	2	10	1
6	1	2	3	2
7	1	2	4	3 (1)
8	1	4	3	1 (1)
12	2	3	7	1
15	2	4	9	1
4	1	0	3	1
5	1	0	4	3 (2)
6	1	0	5	1
7	1	0	6	2
8	1	0	7	1
7	2	0	5	1
9	2	0	7	1
10	2	0	8	2
4	0	1	3	1
7	0	1	6	1
6	0	2	4	1
7.2 (2.1) d	1.3 (0.5) d	1.0 (0.6) d	4.9 (1.8) d	116 e

^aN(if), total number of intrafusal fibers in each muscle spindle.

^bMuscle spindles in the C region were not included because the full complement of fibers is usually present only in the A and B regions.

^cIn parentheses are the number of muscle spindles in complex forms.

^dMean (SD).

^eTotal number of muscle spindles.

One bag fiber with unusual mATPase staining was encountered. This bag fiber displayed exactly the same staining features as bag₁ fibers in the intracapsular region (Figures 5A, 5B, 5D, 5E, 5G, and 5H). In the extracapsular region, however, this fiber stained like a bag₂ fiber (Figures 5C, 5F, and 5I) and it was therefore called “mixed bag₁/bag₂“ fiber.

Immunoreactivity

The staining patterns observed in the A, B, and C regions of nuclear bag₁, bag₂, and chain fibers with a battery of nine antibodies are summarized in Figure 6.

Nuclear Bag₁ Fibers

All nuclear bag₁ fibers were strongly stained with anti-MyHCsto (Figures 5J–5L) and showed no staining with anti-MyHCfet (Figure 7M–7O) along their entire length. The vast majority of bag₁ fibers showed similar staining patterns with the two anti-MyHCI antibodies. Their staining intensity with both anti-MyHCI/1st and anti-MyHCI/2nd MAbs gradually increased along the fiber length from the A to the C region (Figures 7A–7F). Bag₁ fibers were weakly stained with anti-MyHCI/3rd+IIa^∗ only in the outer B and C regions (Figures 7G–7I). Clearly, bag₁ fibers reacted with anti-MyHCIIa antibody (Figure 7J–7L). The number of positive fibers was much higher in the A and B regions than that in the C region, in which only three of 31 bag₁ fibers were stained. Bag₁ fibers usually showed no staining with anti-MyHC“all except IIx” in the equatorial region, and they were either unstained or weakly stained in the juxtequatorial region. Their staining intensity gradually increased along the fiber length from the B into the C region (Figures 5M–5O). The majority of the bag₁ fibers showed weak to moderate staining with anti-MyHCemb in the A region, fewer fibers were stained in the B region, and only 29% of the fibers were stained in the C region (Figures 5P–5R). Approximately one half of the bag₁ fibers in the A region and 16% in the B region were labeled by anti-MyHCα-c, whereas they showed no staining in the C region (Figures 7P–7R).

Nuclear Bag₂ Fibers

Nuclear bag₂ fibers were strongly and evenly stained with anti-MyHCsto along their entire fiber length (Figures 5J–5L) except for three fibers that showed less reactivity at their tapering ending in the C region. Bag₂ fibers were strongly and homogeneously stained with anti-MyHCI/1st and anti-MyHCI/2nd in both the C and B regions (Figure 7B, 7C, 7E, and 7F). In the A region, 97% and 94% of the bag₂ fibers were stained with anti-MyHCI/1st (Figure 7A) and anti-MyHCI/2nd (Figure 7D), respectively. In general, bag₂ fibers were unlabeled by anti-MyHCI/3rd+IIa^∗ in the inner A region and weakly labeled along the remaining of their length (Figures 7G–7I). Although bag₂ fibers mostly did not react with anti-MyHCIIa, a number of fibers were weakly or very weakly stained in the A and B regions (Figures 7J–7L). The staining intensity of bag₂ fibers with anti-MyHC“all except IIx” gradually increased along the fiber length from the A to the C regions (Figures 5M–5O). Bag₂ fibers did not react with anti-MyHCfet in the C region but 15% of these fibers were stained in both the A and B regions (Figures 7M–7O). The majority of bag₂ fibers in the A and B regions were weakly or moderately stained with anti-MyHCemb (Figures 5P–5R). Bag₂ fibers generally showed a higher level of reactivity with anti-MyHCα-c than with bag₁ fibers in the same region (Figures 7P–7R).

Figure 1

Identification of three types of muscle spindles. Serial transverse sections through intracapsular regions of a b₁b₂c spindle (A–C), b₁c spindle (D–F), and b₂c spindle (G,H,I) stained for mATPase at pH 10.4 (A,D,G), pH 4.6 (B,E,H), and pH 4.3 (C,F,J). b₁, bag₁ fiber; b₂, bag₂, fiber. Chain fibers are not marked. Bar = 10 μm.

Nuclear Chain Fibers

The nuclear chain fibers showed no staining with anti-MyHCsto, anti-MyHCI/1st, anti-MyHCI/2nd, and anti-MyHCα-c but were strongly stained with anti-MyHC“all except IIx” (Figures 5 and 7). Almost all chain fibers showed strong staining with anti-MyHCI/3rd+IIa^∗ (Figures 7G–7I) and anti-MyHCIIa (Figures 7J–7L). The chain fibers that were less stained with anti-MyHCI/3rd+IIa^∗ showed also absent or weak staining with anti-MyHCIIa, except in one spindle where two chain fibers were weakly stained by anti-MyHCI/3rd+IIa^∗ but strongly stained by anti-MyHCIIa. Nineteen percent of chain fibers in the A region, 47% in the B region and 64% in the C region showed no staining with anti-MyHCfet, whereas the remainder generally showed decreasing levels of staining intensity from the A to C regions (Figures 7M–7O). Chain fibers showed much higher staining intensity to anti-MyHCemb than bag fibers, although a few of chain fibers in all three regions were negative (Figures 5P–5R). As a general rule, fibers negative to anti-MyHCemb were also negative to anti-MyHCfet.

Exceptions

Although most intrafusal fibers clearly fitted into one of the three fiber types with the general staining patterns described above, two atypical intrafusal fibers were encountered in one spindle. These two fibers, seen in the A region, were similar to chain fibers in diameter and showed low mATPase activity at pH 10.4 and high activity at pH 4.3. They showed no staining with anti-MyHCfet, weak staining with anti-MyHCsto, and moderate to strong staining by all the other MAbs used in the present study (Figure 8).

Figure 2

Dramatic changes in diameter along the length of a bag₂ fiber (arrowhead). Serial cross-sections through intracapsular (A–C) and extracapsular (D) regions stained for mATPase after preincubation at pH 4.6. b₁, bag₁ fiber. Bar = 5 μm.

Discussion

The major highlights provided by the present study regarding the spindles in human deep neck muscles can be summarized as follows: (a) some spindles lacked bag₂ fibers; (b) there was a low incidence of spindles with a unique allotment of intrafusal fiber types; (c) there was a low number of intrafusal fibers per spindle; (d) the content of MyHCemb and MyHCfet varied along the length of nuclear chain fibers; and (e) there were wide variations in MyHC composition among intrafusal fibers.

One-bag-fiber Spindles

Three categories of muscle spindles, b₁b₂c, b₁c, and b₂c, were identified. Our findings show that human neck muscle spindles can lack either bag₁ or bag₂ fibers. Moreover, 10 of 12 b₁c spindles existed as single isolated receptors, indicating that the b₁c spindles in human neck muscles are in fact rather common and are not likely to represent a developmental deviation. The ability to distinguish between b₁c and b₂c spindles relies on the precise classification of the two types of bag fibers. In the outer A and B regions, the bag₂ fiber can be clearly distinguished from the bag₁ fiber by its higher staining intensity after preincubation at pH 4.6 and pH 4.3 (Figure 1). Moreover, immunocytochemistry can also be used as an additional way of identifying intrafusal fiber types, and therefore the use of both these criteria in the present study allowed a reliable classification of nuclear bag₁ and bag₂ fibers.

Figure 3

Three extrafusal muscle fibers (asterisk, A) become enclosed by the spindle capsule. Note that there is a thin inner capsule sleeve surrounding these fibers, which is no longer seen in C. Consecutive sections through different regions of a spindle treated with anti-MyHCemb (A), anti-MyHCsto (B), and anti-MyHCfet (C). The reactivity of the three extrafusal muscle fibers with the antibodies against different MyHC isoforms was similar to that of other ordinary type I extrafusal fibers. Nuclear bag₁ fibers are marked with thick arrows, chain fibers with thin arrows, and bag₂ fibers with arrowheads. Bar = 10 μm.

Figure 4

Transient disappearance of intrafusal fibers. Serial sections taken through the juxtaequatorial (A,B), polar (C–F), and extracapsular (G,H) regions of a muscle spindle, showing the mATPase activity at pH 4.6 (A,C,E,G) and stained with anti-MyHCsto (B,D,F,H). The two bag₁ fibers (b) readily seen in A,B and C,D transiently disappear in E and F but are again seen in the extracapsular region (G,H). c, chain fiber. Bar = 10 μm.

The absence of bag₂ fibers has previously been reported in a small number of muscle spindles in human biceps brachii muscle (Liu et al. 2002). Because of the lack of relevant physiological data on such b₁c spindles in humans, their functional significance remains unknown. However, we can speculate that spindles lacking bag₂ fibers might have a relatively higher dynamic sensitivity, and they might reflect adaptation of the fusimotor system to the particular task of controlling the head posture and movements.

One-bag-fiber spindles have been described in the cat (Banks et al. 1979; Kucera 1982; Kucera and Walro 1987), particularly in the neck muscles (Richmond and Abrahams 1975; Bakker and Richmond 1981,1982; Richmond et al. 1986; Abrahams and Richmond 1988). However, all the one-bag-fiber spindles in cat muscles selectively lack bag₁ fibers (with one reported exception; Kucera 1982), usually appear as part of spindle complexes (Richmond and Abrahams 1979; Bakker and Richmond 1981,1982; Richmond et al. 1986; Abrahams and Richmond 1988), and are restricted to the regions near tendons or tendinous inscriptions in dorsal neck muscles (Bakker and Richmond 1981). We did not find any preferential location for the b₁c spindles in our samples of the human deep neck muscles.

Intrafusal Fiber Type and Number

We have previously shown that each muscle spindle in the human biceps brachii has a unique identity, revealed in part by a unique allotment of numbers of bag₁, bag₂, and chain fibers (Liu et al. 2002). In the human first lumbrical muscle, which is a short muscle with high spindle density, the muscle spindles also have unique combinations of nuclear bag₁, bag₂, and chain fibers (Soukup et al. 2003). However, the present study revealed that only 17% of the muscle spindles in the deep muscles of the neck had unique intrafusal fiber complements, and as many as 18% of the muscle spindles studied in the A and B regions contained one bag₁, one bag₂, and four chain fibers. We compared our previous results with regard to the intrafusal fiber content from human biceps brachii (Liu et al. 2002) and the first lumbrical muscles (Soukup et al. 2003) with the deep neck muscles and found statistically significant differences. Muscle spindles in the lumbrical muscle have the highest number of intrafusal fibers, whereas those in the deep neck muscles contain the lowest fiber number (Figure 9). These differences were mainly due to a lower number of bag₁ fibers in deep neck muscles compared to biceps brachii, and a lower number of bag₁ and bag₂ fibers in biceps compared to lumbrical muscle. The difference between the first lumbrical and the deep neck muscles was due to variation in the number of all three intrafusal fiber types.

Figure 5

mATPase and immunocytochemical staining features of intrafusal fibers. Serial cross-sections of a spindle stained for the demonstration of mATPase at pH 10.4 (A–C), 4.6 (D–F), 4.3 (G–I), and stained with antibodies against MyHCsto (J–L), MyHC“all except IIx” (M–O), and MyHCemb (P–R). Selected sections are shown in the A (left column), B (middle column), and C (right column) regions. Nuclear bag₁ fibers are marked with thick arrows, chain fibers with thin arrows, and bag₂ fibers with arrowheads. Note that one “mix” bag fiber (asterisk) stained as bag₁ in A and B regions and as bag₂ in the C region (F,I). The muscle spindle nerve is seen entering the equatorial region in P. Bar = 20 μm.

Figure 6

Schematic summary of the staining patterns of nuclear bag₁, bag₂, and chain fibers. The relative proportion (%) of each fiber type displaying the different staining levels in the A, B and C regions is shown. Black, strong staining; dark gray, moderate staining; light gray, weak staining; white, no staining.

MyHC Composition in Intrafusal Fibers

This is, to the best of our knowledge, the first investigation to examine the MyHC composition of intrafusal fibers in the deep muscles of the human neck. The immunocytochemical staining profiles of the intrafusal fibers reported here generally paralleled those of human biceps brachii (Liu et al. 2002), although with important exceptions.

The most striking difference was the variation in MyHCemb and MyHCfet content of nuclear chain fibers. The distribution of the embryonic and fetal myosin varied between the chain fibers from different spindles, and along the length of a given chain fiber. These variations seemed to correlate with the variability in mATPase activity noted along the length of individual nuclear chain fibers in the neck muscle spindles. Nuclear chain fibers usually have uniform mATPase activity and a homogeneous MyHC composition along their entire length in muscle spindles of human biceps brachii (Liu et al. 2002) and the first lumbrical muscles (Soukup et al. unpublished data).

There were wide variations in MyHC composition among the intrafusal fibers studied. In fact, we found more variation in the staining patterns along the intrafusal fibers in the deep neck muscles than in the biceps brachii, as shown in Figure 6. For example, staining with MAb A4.840 did generally increase along the length of nuclear bag₁ fibers, but the transition in staining level was spread along the A and B regions. The widely variable staining features revealed in the present study are likely to reflect a wider range of contractile properties of the intrafusal fibers and the more complex architecture and functions of these small deep neck muscles. The deep neck muscles have limited lever action because of their small size (Kamibayashi and Richmond 1998), and in the rhesus monkey they are preferentially recruited during small turns, whereas larger, multiarticular muscles also became active during larger faster turns (Corneil et al. 2001).

Figure 7

MyHC expression in different regions. Serial cross-sections of one spindle stained with anti-MyHCI/1st (A–C), anti-MyHCI/2nd (D–F), anti-MyHCI/3rd+IIa^∗ (G–I), anti-MyHCIIa (J–L), anti-MyHCfet (M–O), and anti-MyHCα-c (P–R). Sections are shown in the A (left column), B (middle column), outer B (right column O,R), and C (right column C,F,I,L) regions, respectively. The intrafusal bundle starts to be encapsulated in section O. Arrows as in Figure 5. Bar = 20 μm.

Figure 8

Intrafusal fibers with unusual staining features. Note that the two fibers marked with open arrowheads stained as nuclear bag₂ fibers with anti-MyHCI/1st (A) but as nuclear chain fibers with both anti-MyHCI/3rd+IIa^∗ (B) and anti-MyHCIIa (C), although they also were labeled with anti-MyHCsto (D). These two fibers were separated from each other by the inner capsule in section E and gradually disappeared. In addition, a new bag₂ fiber (asterisk; G,H) appeared in the B region. Nuclear bag₁, nuclear bag₂, and nuclear chain fibers are marked as in Figure 5. Bar = 10 μm.

The high muscle spindle density and the special features of the muscle spindles in the deep neck muscles allow not only great precision of movement but also adequate proprioceptive information needed both for control of head position and movements and for eye/head movement coordination (Bakker et al. 1984; Edney and Porter 1986; Porter 1986). This study clearly revealed that the muscle spindles of the deep neck muscles have distinct morphological features and MyHC composition. Our data further support the concept that each muscle is unique in both fiber type composition and sensorimotor organization.

Figure 9

Comparison of intrafusal fiber complement (mean ± SD) between human first lumbrical (lum), biceps brachii (bic), and deep neck (dne) muscles. The asterisk indicates statistically significant difference from the first lumbrical muscle and “+” indicates statistically significant difference from biceps brachii. p values are given in parenthesis. ^∗∗, ++ p<0.0001. if indicates all intrafusal fibers.

Footnotes

Acknowledgements

Supported by grants from the Medical Faculty of Umeå University and by The Swedish Research Council.

We thank Drs N. Rubinstein, D.A. Fischman, G.S. Butler-Browne, J.J. Leger, and S. Schiaffino for kindly providing antibodies. The excellent technical assistance of Margaretha Enerstedt is also acknowledged.

References

Abrahams

Richmond

(1988) Specialization of sensorimotor organization in the neck muscle system. Prog Brain Res 76:125–135.

Bakker

Richmond

(1981) Two types of muscle spindles in cat neck muscles: a histochemical study of intrafusal fiber composition. J Neurophysiol 45:973–986.

Bakker

Richmond

(1982) Muscle spindle complexes in muscles around upper cervical vertebrae in the cat. J Neurophysiol 48:62–74.

Bakker

Richmond

Abrahams

(1984) Central projections from cat suboccipital muscles: a study using transganglionic transport of horseradish peroxidase. J Comp Neurol 228:409–421.

Banks

Barker

Stacey

(1979) Sensory innervation of cat hind-limb muscle spindles. J Physiol 293:40P–41P.

Barker

Banks

(1994) The muscle spindle. In Engel

Franzini—Armstrong

, eds. Myology. New York, McGraw-Hill, 333–360.

Bottinelli

(2001) Functional heterogeneity of mammalian single muscle fibres: do myosin isoforms tell the whole story?. Pflugers Arch 443:6–17.

Bottinelli

Reggiani

(2000) Human skeletal muscle fibres: molecular and functional diversity. Prog Biophys Mol Biol 73:195–262.

Cooper

Daniel

(1963) Muscle spindles in man: their morphology in the lumbricals and the deep muscles of the neck. Brain 86:563–586.

10.

Corneil

Olivier

Richmond

Loeb

Munoz

(2001) Neck muscles in the rhesus monkey. II. Electromyographic patterns of activation underlying postures and movements. J Neurophysiol 86:1729–1749.

11.

Djupsjobacka

Johansson

Bergenheim

Wenngren

(1995) Influences on the gamma-muscle spindle system from muscle afferents stimulated by increased intramuscular concentrations of bradykinin and 5-HT. Neurosci Res 22:325–333.

12.

Dubowitz

(1985) Muscle biopsy: A Practical Approach. 2nd ed. London, Bailliere Tindall.

13.

Ecob—Prince

Hill

Brown

(1989) Immunocytochemical demonstration of myosin heavy chain expression in human muscle. J Neurol Sci 91:71–78.

14.

Edney

Porter

(1986) Neck muscle afferent projections to the brainstem of the monkey: implications for the neural control of gaze. J Comp Neurol 250:389–398.

15.

Eriksson

Butler-Browne

Thornell

(1994) Immunohistochemical characterization of human masseter muscle spindles. Muscle Nerve 17:31–41.

16.

Eriksson

Thornell

(1987) Relation to extrafusal fibre-type composition in muscle-spindle structure and location in the human masseter muscle. Arch Oral Biol 32:483–491.

17.

Eriksson

Thornell

(1990) Variation in histochemical enzyme profile and diameter along human masseter intrafusal muscle fibers. Anat Rec 226:168–176.

18.

Ernfors

Lee

Kucera

Jaenisch

(1994) Lack of neurotrophin-3 leads to deficiencies in the peripheral nervous system and loss of limb proprioceptive afferents. Cell 77:503–512.

19.

Gambke

Rubinstein

(1984) A monoclonal antibody to the embryonic myosin heavy chain of rat skeletal muscle. J Biol Chem 259:12092–12100.

20.

Hughes

Cho

Karsch—Mizrachi

Travis

Silberstein

Leinwand

Blau

(1993) Three slow myosin heavy chains sequentially expressed in developing mammalian skeletal muscle. Dev Biol 158:183–199.

21.

Johansson

Sjolander

Djupsjobacka

Bergenheim

Pedersen

(1999) Pathophysiological mechanisms behind work-related muscle pain syndromes. Am J Ind Med Suppl 1:104–106.

22.

Johansson

Sojka

(1991) Pathophysiological mechanisms involved in genesis and spread of muscular tension in occupational muscle pain and in chronic musculoskeletal pain syndromes: a hypothesis. Med Hypotheses 35:196–203.

23.

Kamibayashi

Richmond

(1998) Morphometry of human neck muscles. Spine 23:1314–1323.

24.

Kucera

(1982) One-bag-fiber muscle spindles in tenuissimus muscles of the cat. Histochemistry 76:315–328.

25.

Kucera

Walro

(1987) Heterogeneity of spindle units in the cat tenuissimus muscle. Am J Anat 178:269–278.

26.

Kulkarni

Chandy

Babu

(2001) Quantitative study of muscle spindles in suboccipital muscles of human foetuses. Neurol India 49:355–359.

27.

Larsson

Moss

(1993) Maximum velocity of shortening in relation to myosin isoform composition in single fibres from human skeletal muscles. J Physiol (Lond) 472:595–614.

28.

Leger

Bouvagnet

Pau

Roncucci

Leger

(1985) Levels of ventricular myosin fragments in human sera after myocardial infarction, determined with monoclonal antibodies to myosin heavy chains. Eur J Clin Invest 15:422–429.

29.

Liu

Eriksson

Thornell

Pedrosa—Domellof

(2002) Myosin heavy chain composition of muscle spindles in human biceps brachii. J Histochem Cytochem 50:171–184.

30.

Matre

Sinkjaer

Svensson

Arendt—Nielsen

(1998) Experimental muscle pain increases the human stretch reflex. Pain 75:331–339.

31.

McPartland

Brodeur

Hallgren

(1997) Chronic neck pain, standing balance, and suboccipital muscle atrophy—a pilot study. J Manipulative Physiol Ther 20:24–29.

32.

Ovalle

Smith

(1972) Histochemical identification of three types of intrafusal muscle fibers in the cat and monkey based on the myosin ATPase reaction. Can J Physiol Pharmacol 50:195–202.

33.

Peck

Buxton

Nitz

(1984) A comparison of spindle concentrations in large and small muscles acting in parallel combinations. J Morphol 180:243–252.

34.

Porter

(1986) Brainstem terminations of extraocular muscle primary afferent neurons in the monkey. J Comp Neurol 247:133–143.

35.

Richmond

Abrahams

(1975) Morphology and distribution of muscle spindles in dorsal muscles of the cat neck. J Neurophysiol 38:1322–1339.

36.

Richmond

Abrahams

(1979) What are the proprioceptors of the neck?. Prog Brain Res 50:245–254.

37.

Richmond

Bakker

Stacey

(1986) The innervation of tandem muscle spindles in the cat neck. J Comp Neurol 245:483–497.

38.

Sawchak

Leung

Shafiq

(1985) Characterization of a monoclonal antibody to myosin specific for mammalian and human type II muscle fibers. J Neurol Sci 69:247–254.

39.

Schiaffino

Gorza

Sartore

Saggin

Ausoni

Vianello

Gundersen

(1989) Three myosin heavy chain isoforms in type 2 skeletal muscle fibres. J Muscle Res Cell Motil 10:197–205.

40.

Soukup

Pedrosa—Domellof

Thornell

(2003) Intrafusal fiber type composition of muscle spindles in the first human lumbrical muscle. Acta Neuropathol (Berl) 105:18–24.

41.

Sternberger

(1979) Immunohistochemistry. 2nd ed. New York, Wiley.

42.

Thornell

Grove

Pedrosa

Butler—Browne

Dhoot

Fischman

(1989) Expression of slow tonic myosin in muscle spindle fibers early in mammalian development. In Stockdale

Kedes

, eds. Cellular and Molecular Biology of Muscle Development. New York, Alan R Liss, 471–480.

43.

Walro

Kucera

(1999) Why adult mammalian intrafusal and extrafusal fibers contain different myosin heavy-chain isoforms. Trends Neurosci 22:180–184.

Muscle Spindles in the Deep Muscles of the Human Neck: A Morphological and Immunocytochemical Study

Abstract

Keywords

Materials and Methods

Antibodies and Labeling

Immunocytochemistry

Survey

Statistical Analyses

Results

General Morphological Features

mATPase Activity

Immunoreactivity

Nuclear Bag1 Fibers

Nuclear Bag2 Fibers

Nuclear Chain Fibers

Exceptions

Discussion

One-bag-fiber Spindles

Intrafusal Fiber Type and Number

MyHC Composition in Intrafusal Fibers

Footnotes

Acknowledgements

References

Nuclear Bag₁ Fibers

Nuclear Bag₂ Fibers