Zool. J . Linn. Soc., 50, p p . 101-109. With 1 plate and 1 figure
February 1971
The structure and function of the jaw muscles in the rat (Rattus
norvegicus L.)
11. Their fibre type and composition
KAREN HIIEMAE
Unit of Anatomy with Special Relation to Dentistry, Anatomy Department,
Guy’s Hospital Medical School, London, S.E.l
Accepted for publication April 1970
‘Tonic’ and ‘phasic’ muscle fibre types can be distinguished histologically, using either
histochemical techniques or by staining for lipid with Sudan black B. As muscles of mastication
not only move the lower jaw of the rat, a ‘phasic’ action, but also suspend it from the cranium,
a ‘tonic’ activity, some indication of the contribution of the major muscles to these functions
has been gained from an examination of the fibre content of transverse frozen sections stained
with Sudan black B. The numbers of ‘pale’ (‘phasic’) and ‘dark’ (‘tonic’) fibres were counted
using a montage at a magnification of 60. Results suggest that the anterior temporal, deep
masseter and external pterygoid have an important tonic action in stabilizing the position of
the lower jaw as well as contributing to the production of movement; and that the superficial
masseter and posterior temporal, in particular, have an almost completely phasic action.
These conclusions are entirely consistent with the probable functions of the muscles inferred
from their anatomy.
CONTENTS
.
Introduction
Materials and methods
.
Results .
The distribution of pale and dark fibres
The differential fibre count .
Discussion
.
Acknowledgements .
References
.
.
.
.
PAGE
101
103
104
104
.
.
. 105
. 107
. 108
.
108
INTRODUCTION
The muscles of mastication in mammals can be regarded as having two functions:
to stabilize the mandibular joints, a tonic activity; and to move the mandible, a
phasic action. This is not to imply that these two functions are mutually exclusive,
the reverse is the case. However, the anatomy of the muscles suggests that the anterior
temporal and deep masseter may have a major role in stabilization whilst the posterior
temporal and the superficial masseter are mainly active in moving the lower jaw
(Hiiemae & Houston, 1971). This hypothesis is difficult to test without extensive
physiological investigation into the neuromuscular behaviour of the various parts of
101
102
K. HIIEMAE
the muscles. There is, however, an accumulation of evidence (see Hiiemae, 1966)
which suggests that in mammals different types of striated muscle fibre mediate these
fundions and that these fibre types may be discernible in histological preparations.
Red ‘slow’ fibres have been shown experimentally to be capable of the slow sustained
contraction required for the maintenance of ‘tone’, and white ‘fast’, rapidly contracting
fibres as responsible for phasic action. Most of the available evidence for this correlation between fibre type and function has been obtained from studies of the limb
musculature (e.g. Denny-Brown, 1929) but there is no reason to assume that the
basic distinction between tonic and phasic fibres is absent in the muscles of mastication
(see Cooper, 1960).
Although the jaw muscles are unlikely to show the gross separation of contractile
activity seen in the lower limb where the soleus consists of dark fibres only, their
position and size suggest that there will be an uneven distribution of ‘tonic’ and
‘phasic’ activity and that this may well be reflected by the type and proportion of their
constituent fibres.
Although there are a number of physiological and histochemical criteria available
for the identification of each type of fibre, it must be emphasized that at present there
is no reliable histological guide to the actual speed of contraction of a striated muscle
fibre. Stein & Padykula (1962) examined the physiologically phasic, pale gastrocnemius of the rat and, on the basis of their histochemically determined mitochondrial
enzyme activity, identified three types of fibre: Type A had few mitochondria and
were the classical pale fibres; type B and type C were both red fibres with more
mitochondria, the last being richest in these organelles. Significantly, the tonic
soleus contained only fibres of types B and C. These observations are entirely consistent with those made by Denny-Brown (1929) in his classical study. He found that the
soleus was always composed of at least 95% of slowly contracting fibres and that,
providing the animals (cats) were well fed, the fibres of soleus showed a marked
granularity with considerable sudanophilia. I n contrast, the pale fibres of the
gastrocnemius contracted rapidly and were much less granular with a low affnity for
lipid stains. The correlation between fat content, as indicated by the degree of
sudanophilia present, and the numbers of mitochondria in each type of fibre was
established by Padykula & Gauthier (1963). It appears that the ‘pale’ fibres identified
by the Sudan black B technique correspond with Stein & Padykula’s type A, whilst
the ‘dark’ fibres include both types B and C.
Almost all studies of the relationship between the histological appearance of
striated muscle and its contractile behaviour in mammals have used the limb muscles,
particularly soleus and gastrocnemius. There have, as far as the author is aware, been
no comparable studies of the muscles of mastication. This may be due to their
complexity but if, as seems probable, the jaw muscles have both tonic and phasic
actions, then their anatomy in the rat makes it unlikely that the contribution of each
muscle to both activities will be equal. If this is the case, such differences should be
reflected in the proportions of each type of fibre in each muscle.
Sherrington (1894) attributed the maintenance of muscle tone to the action of
muscle spindles. Cooper (1960) points out that there may be differences in the
structure and distribution of spindles in tonic and phasic muscles; this author found
FIBRE TYPE & COMPOSITION OF JAW MWSCLES IN THE RAT
103
that the numbers of spindles in tonic limb muscles are greater than in the phasic
muscles and that the spindles tend to have a complex form. Cooper also considers
that functionally the jaw muscles closely resemble the limb muscles and therefore the
similarity should extend to the structure of their extrafusal fibres and spindles.
However, in terms of numbers of spindles, the jaw muscles show certain unusual
features. No spindles have been found in the external pterygoid of man (Freimann,
1954; Voss, 1956), the goat, the cat (Cooper, 1960), or the rat (Karlsen, 1965). Voss
(1956) found no spindles in the posterior belly of digastric, few in its anterior belly and
none in the mylohyoid. Karlsen (1965) carefully examined the muscles of mastication
in the rat for the numbers, distribution and type of spindles and for the position and
arrangement of motor endplates. His findings are of interest in view of Cooper’s
(1960) suggestion and the hypothesis that the major tonic muscles in the rat are the
anterior temporal and deep masseter. Karlsen gives the absolute spindle count of
130 for the masseter (taking superficial and deep masseter together), but all except six
were located in the deep masseter. The lateral part of the temporal (posterior temporal
and the outer superficial part of the anterior temporal) had no spindles but the larger
medial, part of the anterior temporal contained 46. This author found 16 spindles in
the internal pterygoid. Spindles of the complex type (Cooper, 1960) were found in
the masseter and the temporal where they showed a tendency to aggregate in clumps
of two to four with occasionally as many as ten. Although no. specific search for
spindles was attempted in this study, they were in fact observed in the sections and
their position and form noted. The aim of this investigation” was: first, to establish
that the muscles of mastication in the rat contained both pale and dark fibres after
staining with Sudan black B; second, to count the numbers of each type of fibre
present in each muscle; and finally to show whether the proportions of each type
differed significantly between the muscles. If the last proved to be the case, this
information coupled with that drawn from anatomical and biomechanical studies
(Hiiemae, 1971 ; Hiiemae & Houston, 1971) should provide a guide to their possible
function.
MATERIALS AND METHODS
The jaw muscles (temporal, superficial masseter, deep masseter, internal and
external pterygoid, digastric and transverse mandibular) were removed from six
well-nourished male rats after they were killed with ether, weighed (see Hiiemae,
1971) and then stretched on card and fixed in 10% formol-saline. Gastrocnemius
and soleus were removed from one rat and used as staining controls. The muscles were
embedded in gelatine, and 20 pm transverse sections cut in a cryostat at -25°C. from
the region of the ‘physiological cross-section’ (Fick, 1911) as determined by inspection.
The sections were stained with Sudan black B in 70% alcohol and mounted in
glycerine on neutral mounting medium.
The prepared slides were first inspected for nerves and muscle spindles. Three
sections of the same muscle but from different animals were then selected for the
* This investigation was undertaken at the Royal Dental Hospital School of Dental Surgery and
at St. Thomas’s Hospital Medical School, University of London and formed part of a Ph.D. Thesis
submitted to the University in June 1966.
104
K. HIIEMAE
fibre count. Sections fulfilling the following requirements were chosen: little or no
disruption of the muscle fasciculi; even staining with the periphery of each fibre
distinct; and few obliquely sectioned fibres. Oblique sectioning was difficult to avoid
in the larger muscles such as deep masseter. The chosen sections were then
photographed and a montage of each at x60 prepared. A counting grid was ruled
on to the montage and the fibres counted using a standard convention. The number
of palely stained, darkly stained and the total number of fibres was counted and the
percentage of each type calculated. No attempt was made to determine the percentage
distribution of the different fibre types within each section as, with the exception of
the posterior temporal, the pale and dark fibres appeared more or less evenly
distributed both within fasciculi and throughout the sections.
All the fibres in the sections of the anterior and posterior temporal and the deep
masseter were counted and the parts of the deep masseter identified. The ratio of
numbers of fibres types in the various parts of the adductors was found and used to
calculate their weight (see Hiiemae, 1971). All the fibres in the external pterygoid and
digastric were counted but only alternate grid squares for one specimen of superficial
masseter and internal pterygoid. A check count of one section of the internal pterygoid
was made to estimate the counting error. No fibre count was attempted for the
transverse mandibular.
RESULTS
The typical appearance of a fasciculus after staining is shown in Plate 1A. The
darkly and palely stained fibres are quite distinct. A similar picture was found in all
the specimens including the control sections, the only variation being in the numbers
of darkly stained fibres present. The results of the fibre count are shown in Table 1
where the source, side and total fibre count for each muscle is given as well as the
percentage of dark and pale fibres. The recount of a section of Internal pterygoid
indicated that the counting error was of the order of 4%.
Muscle spindles were found in the deep masseter (Plate 1B) and the anterior
temporal. Spindles were not found in the central region of any of the other muscles
examined.
The distribution of pale and darkJibres
The arrangement of the fibres and the size of the fasciculi varied considerably
within each section and between muscles. The peripheral fasciculi of the larger
muscles; the temporal, the superficial and deep masseter and the internal pterygoid,
were smaller and more loosely arranged than those in the centre where the fasciculi
were closely packed with little intervening connective tissue. All the small muscles
had small and poorly defined fasciculi. The sizes of the fibres appeared fairly uniform
although the pale were often larger. The fibres in the posterior temporal appeared
larger than those in the other muscles although arranged in compact fasciculi.
The distribution of pale and dark fibres appeared more or less uniform throughout
all sections except for those of the posterior temporal where almost all the dark fibres
FIBRE TYPE & COMPOSITION OF JAW MUSCLES IN T H E RAT
105
were found clustered in a small area in the central part of the deep (cranial) surface
of the muscle. I n the deep masseter, the incidence of dark fibres gradually increased
from in front (anterior deep and anterior deep, infra-orbital) backwards. In general,
the dark fibres were interspersed among the pale, singly or in groups of two or three
throughout the fasciculi.
The dzfuential$bre count
T h e results of the fibre count are shown in Table 1. T h e range of variation between
the sections counted falls within biologically acceptable limits. No count was made of
the fibres in the transverse mandibular as it proved difficult to orientate satisfactorily
due to its elongated triangular shape and small size. T h e sections examined did,
however, show a large proportion of darkly staining fibres.
Table 1. Differential fibre count of the muscles of mastication. The results of the fibre count
from transverse frozen sections of the jaw muscles. The total count and the counts for pale
and dark fibres are given. These are both shown expressed as a percentage of the total fibre
content
Muscle
%
%
Source
Total no.
fibres
No.
pale
No.
dark
pale
Rat VI L.
Rat I11 R.
R a t V L.
Rat V R.
Rat IV L.
Rat VI L.
Rat V R.
R a t V I R.
Rat IV R.
Rat VI R.
Rat V R.
Rat I11 R.
Rat VI R.
R a t V R.
Rat I11 R.
Rat VI R.
R a t V R.
Rat 111 R.
Rat I11 L.
R a t V L.
Rat IV R.
Rat VI L.
Rat I L.
Rat IV R.
Rat I1 L.
Rat IV L.
R a t V R.
Rat I1 L.
Rat IV L.
Rat I L.
6534
6678
5500
6581
6471
4745
5178
4898
6004
4958
7596
7467
3026
3445
2454
13,711
11,866
11,459
4841
3935
2875
2340
1338
3120
1536
1211
1812
1360
1737
1569
3968
3728
3441
5727
5336
3941
4126
3908
5088
3649
5531
5919
2449
2560
1881
8217
6642
6926
3616
2814
2047
1536
786
1825
1078
850
1328
989
1160
1114
2566
2950
2059
854
1135
804
1052
990
916
1289
2065
1548
579
885
573
5494
5218
4533
1227
1121
827
805
552
1295
458
361
484
371
577
45 5
61
56
62
87
83
83
80
80
84
73
73
79
81
75
77
60
56
60
74
72
71
66
59
59
71
70
73
72
67
71
dark
~
Temporalis anterior
Temporalis posterior
Superficial masseter
Anterior deep masseter
Anterior deep masseter (Lo)
Deep masseter
Internal pterygoid
External pterygoid
Digastric ant. belly
Digastric post. belly
39
44
38
13
17
17
20
20
16
27
27
21
19
25
23
40
44
40
25
28
29
34
41
41
29
30
27
28
33
29
Although all the muscles have a higher pale than dark fibre content, their proportion
varies between the muscles, which appear to fall into three groups: those with a very
K. HIIEMAE
106
high dark fibre content, anterior temporal, deep masseter and external pterygoid;
those with a very low dark fibre content, posterior temporal and superficial masseter;
and a third, intermediate, group including the anterior parts of deep masseter, the
internal pterygoid and the digastric. As the sample involved is small, the x2 test has
been applied to investigate whether their differences have any significance. The
results are as follows.
There is no significant difference between the fibre content of the two bellies of
digastric: they belong to the same population.
There is no difference in the percentage of pale fibres in the two parts of anterior
"5c
55
60
65
70
75
Percentage pole fibres
00
05
90
FIGURE
1. A histogram to show the distribution of the percentage pale fibre content in all the
samples counted, two grouping ranges have been used, the solid line determiningthe histogram
based on the narrower range. The mean value of the percentage pale fibre content for each
muscle, with its range, is shown above for comparison.
ADM, Anterior deep masseter; ADM (i-o), anterior deep masseter infra-orbital part;
Dig (ab), digastric, anterior belly; Dig (pb), digastric, posterior belly; DM, deep masseter;
EP, external pterygoid; IP, internal (medial) pterygoid; SM, superficial masseter; TA,
temporalis anterior; TP,temporalis posterior.
deep masseter, but taken together they are significantly different (P< 0.01) from the
percentage of pale fibres in deep masseter ; superficial masseter does not have a different pale fibre content when compared with anterior deep masseter, but it is significantly
different (P< 0.01) from that of deep masseter.
The percentage of pale fibres in the posterior temporal is significantly different
from that of the anterior temporal (P< 0.01).
Further testing shows that:
The percentage fibres counts from the anterior temporal, deep masseter and
external pterygoid fall into the same range or population.
The anterior deep and anterior deep (infra-orbital) parts of masseter, the internal
pterygoid and the digastric also belong to a similar population.
FIBRE TYPE & COMPOSITION OF JAW MUSCLES I N THE RAT
107
The posterior temporal and superficial masseter are of a similar population which
differs significantly from that including the deep masseter and anterior temporal,
but not from the group including the anterior deep masseter.
Although the muscles with a percentage pale fibre content in excess of 80% do
not belong to a different statistical population from those with a pale fibre content
in the 70-75% range, all these taken together form a distinct group when compared
with the deep masseter, anterior temporal and external pterygoid (P< 0.01).
These findings are illustrated by the histogram in Fig. 1. This has been drawn from
the percentage pale fibre content figures for all the sections in Table 1 using two
different groupings so that the units of each grouping system fall within the other.
Whichever grouping is used, the percentage of pale staining fibres within the muscles
of mastication in the rat has a bimodal distribution. Whether this finding has any
functional significance depends on the interpretation of the staining reaction.
DISCUSSION
The technique used in this investigation is simple but subjective as the allocation
of any fibre to the ‘pale’ or ‘dark‘ category depends on visual impression. The staining
reaction results in fibres appearing pale grey, dark grey or black as can be seen in the
photomicrograph (Plate 1A). During counting, ‘pale’ fibres were defined as those
appearing white or pale grey, and ‘dark’ fibres as those stained dark grey or black.
The lack of precision in the staining of the dark fibres may be due to the presence of
two types of fibre, types B and C as identified by Stein & Padykula (1962), each with
a different mitochondria1 enzyme content. If this is the case then all the muscles of
mastication in the rat contain a mixture of fibre types but their degree of heterogeneity
is variable.
The question remains whether any functional significance can be attached to the
fibre content of a muscle as demonstrated by staining with Sudan black B. DennyBrown (1929) showed that the cat Soleus has a slow contraction time and a high
proportion of ‘dark‘ fibres, whereas gastrocnemius had a high predominance of pale
fibres and a rapid but not sustained contraction. Stein & Padykula (1962) found that
the rat soleus contains only fibres of types B and C but that the gastrocnemius has a
large type A fibre content. The soleus is considered a ‘phasic’ muscle and the
gastrocnemius ‘tonic’. There is no such clear division between the percentage fibre
content or presumably, function, in the muscles of mastication. Their position and
internal architecture of the jaw muscles suggests that each contributes to both
activities ;the purpose of this investigation was to determine whether that contribution
is equal. The results suggest that it is not. If the incidence of dark fibres is a guide to
the degree to which a muscle can subserve a tonic or stabilizing function, then those
with a high percentage of dark fibres, deep masseter, anterior temporal and external
pterygoid, are more likely to fulfil this role than the other muscles. This finding is
entirely consistent with their function as suggested by their anatomy (Hiiemae &
Houston, 1971). Equally, the high pale fibre content of the superficial masseter and
posterior temporal suggests that they are probably responsible for rapid movements
of the mandible and are probably ‘phasic’. The remaining muscles probably contribute
108
K. HIIEMAE
both to the production of movement and to the stabilization of the mandible during
the various stages of the gnawing and chewing cycles. (Hiiemae & Ardran, 1968.)
Again the results of this study conform to the suggestions made as to the function of
these muscles based on the anatomical evidence.
I n view of the rapidity of mandibular movement in the rat (Hiiemae & Ardran,
1968), a distinction between ‘tonic’ and ‘phasic’ activity in the jaw muscles might be
considered specious. However, between periods of active ingestion, mastication and
grooming or other ancillary oral activity, the mandible is at rest. Equally, the finding
that all the muscles have a predominance of pale fibres can be explained by a necessity
for rapid rather than sustained contraction in mastication, particularly during the
power stroke which is of very short duration.
The examination of all the sections for muscle spindles was suggested by the
observation (Cooper, 1960) that spindles in tonic muscles tend to be of the complex
type and occur in greater numbers than in phasic muscles. In this study, where no
specific search was undertaken, spindles of the complex type have been found in the
anterior temporal and the deep masseter. This observation coupled with Karlsen’s
(1965) results goes some way to confirm not only Cooper’s observation but also the
suggestion made here that the anterior temporal and deep masseter in the rat have a
major tonic function in slinging the lower jaw from the cranium.
ACKNOWLEDGEMENTS
I should like to thank the late Professor D. V. Davies of St. Thomas’s Hospital
Medical School and Professor H. J. J. Blackwood for their interest and encouragement
during this investigation, W. J. B. Houston for kindly reading the manuscript and
the Nuftield Foundation for their financial support.
REFERENCES
COOPER,
S., 1960. Muscles spindles and other muscle receptors, in Bourne (Ed.), Structure and function
of muscle, Ch. 11, pp. 381417. New York: Academic Press.
DENNY-BROWN,
D. E., 1929. The histological features of striped muscle in relation to its functional
activity. Proc. R. SOC.,104: 371-411.
FICK,R., 1911. Handbuch der Anatomie und Mechamk der Gelenke: 11. Allegemeine Gelenk-und-Muskel
Mechamk. 111. Spezielle Gelenk-und-Muskel Mechamk., 1910, 1911. Jena.
FRBIMANN, R., 1954. Untersuchungen uber Zahl und Anordnung der Muskelspindeln in der
Kaumuskeln des Menschen. Anat. Anz., 100: 258-264.
HIIEMAE,KAFWT,1966. The structure, development and function of the mandibular joint in the rat.
Unpubld. Ph.D. Thesis, University of London.
HIIEMAE,KAREN,1971. The structure and function of the jaw muscles in the rat (Rattus nowegicw L.).
111. The mechanics of the muscles. Zoo1.J. Linn. SOC.,50: 111-132.
HIIEMAE,KAREN
& ARDRAN,
G. M., 1968. A cinefluorographic study of mandibular movement during
feeding in the rat, (Rattus norvegicus).J. Zool., Lond., 154: 139-154.
HI-,
KAREN& HOUSTON,
W. J. B., 1971. The structure and function of the jaw muscles in the rat
(Rattw nmegicus L.). I. Their anatomy and internal architecture. Zool. J. Linn. SOC.,50: 75-99.
KARISEN, K., 1965. The location of motor endplates and the distribution and histological structure of
muscle spindles in the jaw muscles of the rat. Acta. odont. scand., 23: 521-547.
PAD-,
H. A. & GAUTHIER,
G. F., 1963. Cytochemical studies of adenosine triphosphatases in
skeletal muscle fibres. 3. Cell. Biol., 18: 87-107.
SHERRINGTON,
C. S., 1894. On the anatomical constitution of the nerves of skeletal muscles; with
remarks on recurrent fibres in the ventral spinal nerve root.3. Physiol., Lond., 17: 211-258.
Zool.
r. Linn. Sac., 50 (1971)
K. HIIEMAE
Plate 1
(Facing p . 109)
FIBRE TYPE & COMPOSITION OF JAW MUSCLES IN THE RAT
109
STEIN,
J. M. & PADYKULA,
H. A., 1962. Histochemical classification of individual skeletal muscle fibres
of the rat, Am.3. Anat., 110;103-115.
VOSS,H., 1956. Zahl und Anortung der Muskel Spindeln in den oberen Zungbeinmuskeln, in M.
trapezius and M.latissimus dorsi. Anat. Am.,103: 443446.
EXPLANATION OF PLATE
PLATE
1
A.Transverse section of the internal pterygoid after staining with Sudan black B to show the appearance
of a typical fasciculus. x540.
B. Transverse section of deep masseter to show a group of muscle spindles and two nerves, stained
with Sudan black B. x.540.