Reviewing the Morphology of the Jawclosing Musculature In

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THE ANATOMICAL RECORD 294:915–928 (2011)

Reviewing the Morphology of the Jaw-Closing Musculature in Squirrels, Rats, and Guinea Pigs with Contrast-Enhanced MicroCT

PHILIP G. COX* AND NATHAN JEFFERY Department of Musculo-skeletal Biology, University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool, L69 3GE, UK

ABSTRACT Rodents are defined by their unique masticatory apparatus and are frequently separated into three nonmonophyletic groups—sciuromorphs, hystricomorphs, and myomorphs—based on the morphology of their masticatory muscles. Despite several comprehensive dissections in previous work, inconsistencies persist as to the exact morphology of the rodent jaw-closing musculature, particularly, the masseter. Here, we review the literature and document for the first time the muscle architecture nonin- vasively and in 3D by using iodine-enhanced microCT. Observations and measurements were recorded with reference to images of three individu- als, each belonging to one of the three muscle morphotypes (squirrel, guinea pig, and rat). Results revealed an enlarged superficial masseter muscle in the guinea pig compared with the rat and squirrel, but a reduced deep masseter (possibly indicating reduced efficiency at the incisors). The deep masseter had expanded forward to take an origin on the rostrum and was also separated into anterior and posterior parts in the rat and squirrel. The zygomaticomandibularis muscle was split into anterior and posterior parts in all the three specimens by the masseteric nerve, and in the rat and guinea pig had an additional rostral expansion through the infraorbital foramen. The temporalis muscle was found to be considerably larger in the rat, and its separation into anterior and posterior parts was only evident in the rat and squirrel. The pterygoid muscles were broadly similar in all three specimens, although the internal pterygoid was somewhat enlarged in the guinea pig implying greater lateral movement of the mandible during chewing in this species. Anat Rec, 294:915–928, 2011. VC 2011 Wiley-Liss, Inc.

Key words: microCT; contrast-enhanced; rodent; masseter; temporalis; pterygoid

The masticatory apparatus of rodents is unique among mammals and deﬁnes the order Rodentia. As well as Grant sponsor: Natural Environment Research Council possessing an extremely unusual dentition—a pair of (NERC); Grant number: NE/G001952/1. grossly enlarged incisors separated from the cheek teeth *Correspondence to: Philip G. Cox, Department of Musculo- by a large diastema—rodents have a highly distinctive skeletal Biology, University of Liverpool, Sherrington Buildings, arrangement of masticatory muscles. The masseter is by Ashton Street, Liverpool, L69 3GE, UK. Fax: þ44 151 794 5517. far the dominant jaw-closing muscle in the Rodentia, E-mail: [email protected] comprising between 60% and 80% of the entire mastica- Received 10 February 2011; Accepted 17 February 2011 tory muscle mass (Turnbull, 1970). Furthermore, the DOI 10.1002/ar.21381 musculature has become specialized to accomplish not Published online 28 April 2011 in Wiley Online Library only gnawing at the incisors and chewing at the molars (wileyonlinelibrary.com).

VC 2011 WILEY-LISS, INC. 916 COX AND JEFFERY but also propalinal movement of the lower jaw between dea (pocket gophers, kangaroo rats, and mice). In the these two feeding modes (Becht, 1953). These movements hystricomorph masticatory apparatus, a deeper part of are necessary in rodents, because it is impossible for the the masseter has extended forward, through the orbit incisors and cheek teeth to be in occlusion at the same and the grossly enlarged infraorbital foramen to take an time, and thus, incision and mastication have become origin on the snout. This morphology is found in the mutually exclusive activities (Hiiemae and Ardran, 1968). Caviomorpha (South American rodents), Phiomorpha Given the unique demands on the masticatory apparatus, (African mole-rats, cane rats, and the dassie rat) and it is perhaps unsurprising that the morphology of the Hystricidae (old world porcupines) as well as the previ- jaw-closing muscles, in particular the masseter, has long ously mentioned Pedetidae, Anomaluridae, Dipodidae, been used to classify the rodents into subgroups. and Ctenodactylidae. Finally, the myomorphs combine Brandt (1855) first used features primarily from the sciuromorph and hystricomorph features with the ori- masticatory apparatus to group rodents into squirrel- gins of both parts of the masseter having migrated on to like (Sciuromorpha), mouse-like (Myomorpha), and por- the rostrum. This condition is seen in the Muroidea cupine-like (Hystricomorpha) forms (Brandt’s fourth (mice and rats) and the Gliridae (dormice). group Lagomorpha, the rabbits, hares, and pikas, now The above morphological descriptions have been occupy a separate, albeit closely related, order). These greatly complicated by the complete lack of consensus on three suborders were largely retained with only minor the nomenclature of rodent masticatory muscles, with revisions by most workers for the next century (e.g., particular regard to the masseter (hence the lack of spe- Thomas, 1896; Miller and Gidley, 1918) and indeed were cific muscle nomenclature in the previous paragraph). still the basis for rodent taxonomy in George Gaylord Part of the confusion arises due to the uncertainty of Simpson’s monumental classification of the mammals in how many layers the masseter divides into, and whether 1945. It should be noted, however, that Simpson alluded all of these layers should be referred to as the masseter to a growing dissatisfaction with the three suborders or as entirely separate muscles. This uncertainty per- (Simpson, 1945, p.198) but retained them in his work sists despite several thorough descriptive articles based owing to a lack of a better alternative at that time. The on gross dissection (in particular, compare Hiiemae and problem with the three suborder arrangement can be Houston, 1971 with Weijs, 1973; see also Turnbull, 1970; clearly seen in Simpson’s classification: there are a num- Woods, 1972; Cooper and Schiller 1975; Ball and Roth, ber of rodent families that do not neatly fit into the 1995; Druzinsky, 2010a). A possible cause of much of Sciuromorpha, Myomorpha, or Hystricomorpha. In par- this uncertainty is methodological in that information on ticular, the Anomaluridae (scaly-tailed squirrels), Pedeti- muscle morphology is typically gathered invasively by dae (springhare), Dipodidae (jerboas, jumping mice, and dissection, and thus, superficial structures are destroyed birchmice), Bathyergidae (mole-rats), and Ctenodactyli- in gaining access to the deeper structures and spatial dae (gundis) have all posed problems to various workers relationships are difficult to define accurately. On the in the past. A competing classification of rodents, first basis of the work by Metscher (2009), Jeffery et al. proposed by Tullberg (1899), split the Rodentia into two (2011) recently developed an alternative, nondestructive suborders (Sciurognathi and Hystricognathi) based on approach that has the potential to reveal detailed infor- the morphology of the angular process of the mandible. mation on rodent masticatory muscle morphology in This system overlaps with the masseter-based classifica- situ. In this method, the biological specimen is soaked in tion in some respects, such as the fact that all hys- a solution of iodine for a number of weeks before scan- tricognaths have a hystricomorph muscle arrangement ning, which allows the detailed imaging of soft tissues (Lavocat, 1974; Wood, 1974), but has notable differences and hard tissues. Here, we use this novel technique in as well, particularly that sciurognaths can possess any reviewing the structure, arrangement, and terminology of the three masticatory muscle morphologies (Offer- of the masticatory apparatus in three rodents displaying mans and De Vree, 1989). the sciuromorph, hystricomorph, and myomorph muscle Neither of the two classifications outlined above has arrangements. Descriptions of the masticatory muscles stood the test of time. Although evidence points toward will be reported, comparisons with previous work will be a monophyletic Hystricognathi, the Sciurognathi is made, and current controversies will be addressed. almost certainly a paraphyletic grouping, and the idea Given the importance of rodents and knowledge of their that the three suborders of Brandt (1855) and Simpson musculature to many scientific disciplines other than (1945) represent monophyletic groups of rodents is now anatomy, in particular evolutionary developmental biol- generally discredited (Adkins et al., 2001; Huchon et al., ogy (Hallgrı´msson et al., 2007; Jones et al., 2007; Hall- 2002; Adkins et al., 2003; Blanga-Kanfi et al., 2009). grı´msson and Lieberman, 2008; Parsons et al., 2008), it However, the use of the terms sciuromorph, myomorph, is felt that such a review is timely and necessary. and hystricomorph as adjectives describing particular arrangements of jaw-closing muscles has persisted, MATERIALS AND METHODS largely thanks to Wood (1965). In his work, Wood Sample describes the primitive arrangement of rodent masticatory muscles (the ‘‘protrogomorph’’ condition, found in Three rodent species were chosen that possess the most pre-Oligocene fossil rodents and also in the extant sciuromorph, hystricomorph, and myomorph morpholo- mountain beaver, (Aplodontia rufa), and the three gies. These were, respectively, the Eastern grey squirrel arrangements derived from it. In the sciuromorph condi- (Sciurus carolinensis), the domesticated guinea pig tion, part of the masseter has expanded anterodorsally (Cavia porcellus), and the brown rat (Rattus norvegicus). to take its origin from the rostrum and the widened root These species were selected as they all have been of the zygomatic arch. This arrangement is seen in the well-studied previously, and each represents a typical Sciuridae (squirrels), Castoridae (beavers), and Geomyoi- member of its feeding type (i.e., none is anomalously RODENT MASTICATORY MUSCLES 917

Fig. 1. Coronal lCT slice through head of squirrel. dm, deep masseter; m, mandible; sm, superficial masseter; zm, zygomaticomandibularis. Scale bar: 5 mm. Slice position shown by dashed line. specialized). To select an average individual of each spe- fixed in phosphate-buffered formal saline solution (poly- cies to study, formalin-fixed heads of eight rats, eight merized formaldehyde dissolved as a 4% solution in guinea pigs, and seven squirrels were imaged using phosphate-buffered saline allowing for long term storage microCT. Subsequently, 59 three-dimensional landmarks with limited tissue shrinkage) and then placed in 15 to were taken from the skull and mandible of each speci- 25% I2KI contrast agent for a period of between 2 to 7 men using Amira 5.2 (Mercury Systems, Chelmsford, weeks depending on size. These incubation times were MA). From this data, variation in the shape of the skull insufficient for the passive diffusion throughout the and mandible within each species was analyzed using larger Cavia specimen. Small volumes of contrast agent geometric morphometrics (O’Higgins, 2000; Adams et al., (15%) were therefore injected into the body of the 2004), implemented with the software Morphologika2 muscles with a fine grade needle and incubated for a v2.5 (O’Higgins and Jones, 1998). The landmark co-ordi- further 2 weeks. All three specimens were imaged with nates were subjected to Procrustes superimposition to the Metris X-Tek custom 320 kV bay system at the remove translation, rotation, and size differences, and EPSRC funded Henry Moseley X-ray Imaging Facility, then a principal components analysis was performed for University of Manchester. Imaging parameters were each species. For each specimen, the squared value of optimized for each specimen to maximize spatial and each PC score was multiplied by the percentage of varia- contrast resolution as well as data handling. Voxel reso- tion encapsulated by that principal component, and the lutions varied from 0.033 to 0.040 mm. results were summed over the first seven principal components. The specimen with the lowest total within each species was determined to be the individual closest to the Observations and Reconstructions mean shape of the sample (which is located at the origin The contrast-enhanced microCT images (e.g., Figs. 1–3) of the principal axes, O’Higgins, 2000), and this individ- were viewed using Amira 5.2. The morphology of each ual was used to study the masticatory musculature. jaw-closing muscle was determined and described. Three-dimensional reconstructions of all the jaw-closing muscles were created for each specimen using the seg- Imaging mentation function of Amira 5.2 (e.g., Fig. 4). Recon- To visualize the muscle tissues and the bone in a non- structions of the skull and mandible were also created to destructive manner, the specimen best approximating facilitate visualization of the origins and insertions of the mean shape of the sample of each species was each muscle. As can be seen from the microCT images imaged again, this time using contrast-enhanced (e.g., Figs. 1–3), the difference in contrast between mus- microCT. The enhancement uses a solution of iodine cle and bone was not sufficient to allow the models to be (I2KI) to increase the differential attenuation of X-rays generated using the threshold function. Hence, the mus- among soft tissues and has been shown to demonstrate cle and bone reconstructions were built by manually patterns of muscle fibers and fascicles against the painting the object of interest in a number of slices and connective tissues (Jeffery et al., 2011). Specimens were interpolating between them. A smoothing function was 918 COX AND JEFFERY

Fig. 2. Coronal lCT slice through head of rat. dm, deep masseter; lt, lateral temporalis; m, mandible; mt, medial temporalis; rsm, reflected part of superficial masseter; sm, superficial masseter; zm, zygomaticomandibularis. Scale bar: 5 mm. Slice position shown by dashed line.

Fig. 3. Coronal lCT slice through head of guinea pig. dm, deep masseter; ep, external pterygoid; ip, internal pterygoid; m, mandible; rsm, reflected part of superficial masseter; sm, superficial masseter; t, temporalis; zm, zygomaticomandibularis. Scale bar: 5 mm. Slice position shown by dashed line. used to reduce the blocky appearance of the reconstruc- verted to masses assuming muscle density of 1.0564 g/ tions. Amira 5.2 was also able to output the volume of cm3 (Murphy and Beardsley, 1974). The condylobasal each muscle in each reconstruction. These were con- skull length for each specimen was measured from the RODENT MASTICATORY MUSCLES 919 pus); Satoh and Iwaku, 2006 (Onychomys); Satoh and Iwaku, 2009 (Neotoma, Peromyscus); Schumacher and Rehmer, 1962 (Cavia, Rattus); Thorington and Darrow, 1996 (Aplodontia, Paraxerus, Funisciurus, Myosciurus, Heliosciurus, Protoxerus, Funambulus, Calliosciurus, Tamiops, Xerus, Atlantoxerus, Ratufa); Turnbull, 1970 (Scuirus, Rattus, Hystrix); Weijs, 1973 (Rattus); Wood, 1965 (Marmota, Myocastor, Ondatra); Wood and White, 1950 (Chinchilla); Woods, 1972 (Proechimys, Echimys, Isothrix, Mesomys, Myocastor, Octodon, Ctenomys, Ere- thizon, Cavia, Chinchilla, Dasyprocta, Thryonomys, Pet- romus); Woods and Howland, 1979 (Capromys, Geocapromys, Plagiodontia, Myocastor); Woods and Her- manson, 1985 (Capromys, Geocapromys, Plagiodontia, Myocastor, Echimys, Octodon, Erethizon, Coendou, Dasy- procta, Atherurus, Thryonomys, Petromus).

Nomenclature In this article, the three layers of the masseter will be referred to as the superficial masseter, the deep masseter, and the zygomaticomandibularis (following Turn- bull, 1970; Weijs, 1973; Ball and Roth, 1995 among others). There are a number of nomenclatures currently in use, all of which have their advantages. The principal competing system, favoured by Wood (1965) and Woods (1972), names the three layers superficial masseter, lateral masseter and medial masseter; but also see Satoh and Iwaku (2004, 2006, 2009) and Druzinsky (2010a,b) who combine different parts of these two nomenclatures. The system used here has been chosen for its consis- tency with the nomenclature used in most other mammalian groups (e.g. Storch, 1968; Coldiron, 1977; Janis, 1983). The rostral expansion of the innermost layer in myomorphs and hystricomorphs, sometimes known as the maxillomandibularis (e.g. Mu¨ ller, 1933; Schumacher and Rehmer, 1962; Turnbull, 1970), is here termed the infraorbital part of the zygomaticomandibularis. Where the temporalis has been split into two parts, they are named the medial and lateral parts, as this is felt to Fig. 4. Left lateral view of 3D reconstructions of the skull, mandible reflect more accurately their anatomical relationship to and masticatory muscles of (A) guinea pig, (B) rat and (C) squirrel. one another than anterior and posterior. The pterygoid adm, anterior deep masseter; iozm, infraorbital part of zygomatico- muscles are referred to as internal and external in refer- mandibularis; lt, lateral temporalis; mt, medial temporalis; pdm, posterior deep masseter; sm, superficial masseter; t, temporalis. All scale ence to their origin in and on the pterygoid fossa. bars: 5 mm. microCT scans. This length was used to estimate body RESULTS mass, using the independent contrasts-corrected formula Table 1 shows the masses of all jaw-closing muscles given by Reynolds (2002). and the relative proportion of the total muscle mass that The observed arrangements of the jaw-closing muscles each comprises for all three specimens. Table 2 gives the were compared with those seen in previous literature, condylobasal skull length and estimated body mass of both of the three species under study and other rodent each specimen to allow comparison with other literature. species. The literature consulted was as follows: Ball Figures 1–3 demonstrate the enhanced microCT imaging and Roth, 1995 (Sciurus, Microsciurus, Sciurillus, of the muscles in the squirrel, rat, and guinea pig, Tamiasciurus, Tamias, Glaucomys); Byrd, 1981 (Cavia); respectively. Regions of light (high X-ray attenuation) Druzinsky, 2010a (Aplondontia, Cynomys, Tamias, Mar- represent groups of muscle fibers (see Jeffery et al., mota, Ratufa, Sciurus, Thomomys); Greene, 1935 (Rat- 2011). Darker bands represent the epimysium and peri- tus); Hiiemae and Houston, 1971 (Rattus); Mu¨ ller, 1933 mysium that separate muscles and fascicles. It can be (Hydrochaerus); Offermans and De Vree, 1989 (Pedetes); seen that although the microCT images of the guinea Olivares et al., 2004 (Aconaemys, Octomys, Tympanoc- pig (Fig. 3) are less well resolved (due in part to the tomys, Spalacopus, Octodon, Octodontomys); Satoh, larger size of the specimen) than those of the squirrel 1997, 1998, 1999 (Apodemus, Clethrionomys); Satoh and and rat (Figs. 1 and 2), the separation between muscle Iwaku, 2004 (Mesocricetus, Cricetulus, Tscherkia, Phodo- layers can still be distinguished. 920 COX AND JEFFERY TABLE 1. Masses and percentages of jaw-closing muscles Squirrel Guinea pig Rat Muscle Mass (g) % Mass (g) % Mass (g) % Superficial masseter 0.123 27.37 0.219 45.35 0.064 20.42 Deep masseter 0.162 36.04 0.037 7.57 0.108 34.34 Anterior 0.099 21.96 0.047 14.91 Posterior 0.063 14.07 0.061 19.43 Zygomaticomandibularis 0.039 8.70 0.071 14.75 0.021 6.76 Anterior 0.030 6.77 0.029 5.95 0.005 1.59 Posterior 0.009 1.93 0.006 1.32 0.003 0.94 Infraorbital 0.036 7.48 0.013 4.24 Temporalis 0.054 12.03 0.051 10.65 0.085 26.86 Lateral 0.006 1.40 0.024 7.73 Medial 0.048 10.63 0.060 19.14 Pterygoid 0.071 15.87 0.105 21.68 0.037 11.62 External 0.019 4.30 0.014 3.00 0.010 3.33 Internal 0.052 11.56 0.090 18.68 0.026 8.29 Total 0.450 100.00 0.483 100.00 0.315 100.00

TABLE 2. Skull length and estimated body mass the front of the zygomatic arch to the tip of the angle, of rodents in this study thus revealing the deep masseter behind. Some workers have divided this muscle into two parts Skull length (mm) Estimated body mass (g) based on the two insertion areas on the lateral and Squirrel 48.22 311 medial surfaces of the mandible (Greene, 1935; Yoshi- Guinea pig 57.51 573 kawa and Suzuki, 1969; Woods, 1972; Woods and How- Rat 43.40 216 land, 1979; Woods and Hermanson, 1985). However, this study, along with many others (e.g., Wood, 1965; Weijs, 1973; Ball and Roth, 1995; Thorington and Darrow, 1996; Druzinsky, 2010a), retains it as a single muscle mass as there is no clear separation seen in the con- Superficial Masseter trast-enhanced microCT images. The outermost layer of the masseter is clearly distinguished from the deeper layers in the microCT images. Deep Masseter There is a clear dark band separating the superficial masseter from the deep masseter in coronal view in all This muscle layer, sandwiched between the superficial three rodents (Figs. 1–3). From the reconstructions (Fig. masseter and zygomaticomandibularis (Fig. 5), takes its 4), it can be seen that the superficial masseter exhibits a origin from the ventrolateral surface of the zygomatic fairly consistent morphology across the three specimens. arch and inserts on the lateral surface of the mandible This muscle takes a small origin from a flattened tendon all along the masseteric ridge from beneath the second attached to a small tubercle or process just below the in- molar to the angular process. The microCT images show fraorbital foramen. The posterodorsal muscle fascicles that this muscle layer clearly divides into two sections, then run to the back of the mandible to insert into a anterior and posterior, in the squirrel and rat (Fig. small region on the posterolateral surface of the angle of 6a,b). Figure 6a also clearly shows the separation of the the jaw ramus. Some of the muscle fascicles on the dor- superficial and deep masseters in axial view, based on sal edge of this muscle also insert on to the aponeurosis variation in the fascicle direction. The anterior part of of the deeper masseteric layer, making the separation of the deep masseter originates from the rostrum and these two parts of the masseter frequently difficult. The inserts on the anterior portion of the masseteric ridge, anteroventral fascicles of the superficial masseter run and the posterior part originates further back on the zy- under the mandible to insert on the medial surface of gomatic arch and inserts on the mandibular angle. Such the jaw in a fossa just ventral to the insertion of the in- a division is not so easily discerned from the microCT ternal pterygoid (Figs. 1–3). In the rat, there is also a images of the guinea pig (Fig. 6c) and so, in this speci- dorsal elongation of this reflected part of the superficial men, the deep masseter has been reconstructed as a sin- masseter anterior to the internal pterygoid (Fig. 2). This gle muscle. In the rat and squirrel, the deep masseter pars reflexa (Turnbull, 1970; Woods, 1972; Weijs, 1973) has spread anteriorly on to the rostrum to originate is even more well-developed in the guinea pig, extending from the masseteric fossa and the widened inferior root as far as the medial condyloid process (Fig. 3). It can be of the zygomatic process of the maxilla, as can be seen seen from Table 1 that the superficial masseter is rela- from the 3D reconstructions (Fig. 7b,c). In the rat, the tively larger in the guinea pig (Fig. 4a) than in the rat origin of the deep masseter extends as far as the ante- or squirrel (Fig. 4b,c), forming almost half of the jaw- rior margin of the maxilla, and in the squirrel, beyond closing musculature. Its dorsal edge is at the level of the this point on to the premaxilla. In the guinea pig (Fig. zygomatic arch, almost completely obscuring the deeper 7a), this muscle is restricted to the zygomatic arch by layers in lateral view. In contrast, the superficial mass- the large infraorbital foramen. Thus, the deep masseter eter of the rat and squirrel is much more restricted dor- is a relatively much smaller muscle in the guinea pig sally, its margin running diagonally from the origin at than in the rat and squirrel, where it forms well over RODENT MASTICATORY MUSCLES 921

Fig. 5. Coronal lCT slice through head of rat. dm, deep masseter; m, mandible; sm, superﬁcial masseter; za, zygomatic arch; zm, zygomaticomandibularis. Scale bar: 5 mm. Slice position shown by dashed line.

Fig. 6. Transverse lCT slices through head of (A) squirrel, (B) rat and (C) guinea pig. adm, anterior deep masseter; azm, anterior zygomaticomandibularis; dm, deep masseter; ep, external pterygoid; iozm, infraorbital part of zygomaticomandibularis; m, mandible; pdm, posterior deep masseter; pzm, posterior zygomaticomandibularis; sm, superﬁcial masseter; t, temporalis. All scale bars: 5 mm. Slice positions shown by dashed lines.

30% of the masticatory muscle volume (see Table 1). tain other researchers, generally working on sciuro- This reduced size and restricted origin may explain its morphs and myomorphs (Yoshikawa and Suzuki, 1969; lack of separation into anterior and posterior parts. Weijs, 1973; Ball and Roth, 1995; Thorington and Dar- The division of the deep masseter of the squirrel and row, 1996; Druzinsky, 2010a). It is less common in stud- rat into anterior and posterior parts is also made by cer- ies of the hystricomorphs where there is no expansion of 922 COX AND JEFFERY also clearly visible as a separate entity in the squirrel (Fig. 8) and guinea pig (Fig. 3). The innermost layer of the masseter runs between the zygomatic arch and the dorsal part of the mandible. More specifically, it originates from the medial surface of the zygomatic arch (not only largely on the jugal but also on the parts of the maxilla and squamosal) and inserts on the lateral surface of the lower jaw. In squirrels (Fig. 8), the insertion is in the masseteric fossa, just posterior to the toothrow and ventral and anterior to the mandibular condyle. In rats (Figs. 2 and 9) and guinea pigs (Fig. 10), the zygomaticomandibularis inserts on the lateral crest below the second and third molars and on to the coronoid process. In particular, the microCT images of the guinea pig (Fig. 10) show very clearly the ventromedial orientation of the muscle fascicles between the zygomatic arch and lower jaw. As in the previous layer, the microCT images provide good evidence to jus- tify splitting this muscle into anterior and posterior parts. The anterior part runs ventrally from the medial surface of the jugal to the lateral crest, whereas the posterior part originates on the ventral and medial surface of the zygomatic process of the squamosal and runs an- teroventrally to the coronoid process of the mandible. As well as showing different orientations of their muscle fascicles, the two parts are also clearly separated by the masseteric nerve (Figs. 8 and 9). In the rat and the guinea pig, there is an anterodorsal expansion of the zygomaticomandibularis into the orbit and through the infraorbital foramen to take an origin from the rostrum. The expansion of this muscle is relatively large in the guinea pig (Fig. 11a), extending through the grossly enlarged infraorbital foramen to take a large origin on the premaxilla as well as on the maxilla. However, the rostral origin is much smaller in the rat (Fig. 11b), restricted to the area of the maxilla dorsal to the masseteric fossa (the origin of the anterior deep masseter). This expansion of the zygomaticomandi- Fig. 7. Left lateral view of 3D reconstructions of the skull, mandible bularis is completely absent in the squirrel (Fig. 11c). In A B C and masticatory muscles of ( ) guinea pig, ( ) rat and ( ) squirrel with both the rat and guinea pig, the infraorbital part of the the superficial masseter and lateral temporalis removed. adm, anterior deep masseter; dm, deep masseter; iozm, infraorbital part of zygoma- zygomaticomandibularis inserts into a fossa at the ante- ticomandibularis; mt, medial temporalis; pdm, posterior deep mass- rior end of the lateral crest, ventrolateral to the first eter; t, temporalis. All scale bars: 5 mm. cheek tooth. Figure 6b shows all three parts of the zygomaticomandibularis of the rat, clearly separated from one another, in axial view. Overall, the zygomaticoman- the muscle on to the rostrum. Woods (1972), working on dibularis makes up a small part of the masticatory mus- a number of hystricomorph genera, splits the masseter culature—less than 10% in the rat and squirrel (see lateralis profundus into anterior and posterior parts but Table 1). It is slightly greater in the guinea pig (15%) notes that the separation is difficult to see in Cavia. owing to the large infraorbital portion in this species. This was certainly the case in this study, the microCT In a few studies (generally on hystricomorph rodents), scans giving no indication of such a division in the deep an extra section of this muscle has been distinguished masseter of the guinea pig. running almost horizontally from the lateral jugal fossa of the zygomatic arch to the postcondyloid process on the mandible. Woods (1972) and Wood (1974) refer to it Zygomaticomandibularis as the ‘‘masseter lateralis profundus, pars posterior, Hiiemae and Houston (1971) do not recognize the deep division’’; subsequent studies (Woods and How- zygomaticomandibularis as a separate muscle in the rat, lands, 1979; Woods and Hermanson, 1985; Offermans finding it insufficiently distinguishable from the deep and De Vree, 1989; Olivares et al., 2004) use the less masseter. This arrangement is followed in more recent cumbersome ‘‘posterior masseter.’’ Druzinsky (2010a) works, such as Byrd (1981), Satoh (1997, 1998, 1999), describes the posterior masseter in Aplodontia rufa and and Bresin et al. (2000). However, the microCT images Marmota monax, but notes that, owing to its more verti- of the rat in this study show a distinct division between cal course in these rodents, it is very difficult to separate the deep masseter and zygomaticomandibularis (Figs. 5 it from the posterior zygomaticomandibularis. No evi- and 6b), and so the latter has been retained as a discrete dence of a separate posterior masseter was found in any muscle (in line with Turnbull, 1970; Weijs, 1973). It is of the contrast-enhanced microCT images used in this RODENT MASTICATORY MUSCLES 923

Fig. 8. Coronal lCT slice through head of squirrel. azm, anterior zygomaticomandibularis; dm, deep masseter; ep, external pterygoid; ip, internal pterygoid; lt, lateral temporalis; m, mandible; mn, mandibular nerve; mt, medial temporalis; pzm, posterior zygomaticomandibularis; sm, superﬁcial masseter; za, zygomatic arch. Scale bar: 5 mm. Slice position shown by dashed line.

Fig. 9. Coronal lCT slice through head of rat. azm, anterior zygomaticomandibularis; dm, deep masseter; ep, external pterygoid; ip, internal pterygoid; lt, lateral temporalis; m, mandible; mn, mandibular nerve; mt, medial temporalis; pzm, posterior zygomaticomandibularis; sm, superﬁcial masseter; za, zygomatic arch. Scale bar: 5 mm. Slice position shown by dashed line. 924 COX AND JEFFERY

Fig. 10. Coronal lCT slice through head of guinea pig. m, mandible, sm, superficial masseter, za, zygomatic arch; zm, zygomaticomandibularis. Scale bar: 5 mm. Slice position shown by dashed line. study, in agreement with Weijs (1973), Cooper and Schil- compared with that of the squirrel and rat (Fig. 12)—a ler (1975), and Ball and Roth (1995). separation of the guinea pig temporalis may yet exist and be visible with higher quality image data. The division of the temporalis into two parts has been Temporalis made by many authors (Woods, 1972; Weijs, 1973; Woods The temporalis muscle is a relatively small muscle, and Howlands, 1979; Ball and Roth, 1995; Thorington compared with the masseter, in the squirrel and guinea and Darrow, 1996; Druzinsky, 2010a). Some previous pig, forming around 10% of muscle mass (see Table 1). research (Woods, 1972; Weijs, 1973) has separated the However, it is a much larger component in the rat, ventral most fibers of the temporalis to create a third di- accounting for over a quarter of jaw-closing musculature vision, the posterior temporalis (called the suprazygo- (as also noted by Schumacher and Rehmer, 1962). This matic by Druzinsky, 2010a). This usually consists of comparison is most clearly illustrated in axial view (see those fibers originating from the caudal region of the Fig. 12), in which the temporalis muscles of the rat are squamosal (Weijs, 1973), or in some cases, just those shown to be at least double in mediolateral width rela- fibers taking origin from the zygomatic process of the tive to skull size.. In rats and squirrels, the microCT squamosal (Woods, 1972; Hautier and Saksiri, 2009; images show a division of the temporalis into medial Druzinsky, 2010a). Hautier (2010) describes a third divi- and lateral parts (Figs. 2 and 8). The medial temporalis sion of the temporalis in Ctenodactylus but suggests that takes its origin from the lateral surface of the cranium, this is not universal among rodents and may result from extending rostrocaudally from the frontal-parietal suture lateral displacement of the temporalis owing to the dis- to the lambdoid crest, and dorsoventrally from the tem- tal position of the eye. Such an eye position and the con- poral ridge to the external auditory meatus and zygo- comitant third division of the temporalis are also found matic process of the squamosal. The muscle fascicles in the dipodoids (Klingener, 1964). This temporal divi- from this wide origin converge to a small region on the sion was not clearly separable in any of the three speci- medial surface of the mandible between the retromolar mens under study here. fossa and the coronoid process. The lateral temporalis takes its origin from the anterior half of the fascia over- Internal Pterygoid lying the medial temporalis and inserts on the coronoid process. It is a relatively large component in the rat, This muscle is well-developed in rodents and has a extending widely over the surface of the medial tempora- similar morphology in all three species in this study. It lis (Fig. 4b) but much more limited in origin in the is particularly notable in the guinea pig in which it squirrel (Fig. 4c). This division between medial and lat- accounts for almost a fifth of the jaw-closing muscula- eral was not visible in the guinea pig microCT images ture (see Table 1 and Fig. 3). The internal pterygoid and so the temporalis has been reconstructed as a single originates in the pterygoid fossa posterior to the molar muscle in this specimen (Fig. 4a). However, the temporal toothrow. In squirrels, it also has an origin on the lateral region of the guinea pig microCT scan is poorly resolved surface of the pterygoid process. From here, the internal RODENT MASTICATORY MUSCLES 925 culature (see Table 1). It originates along the ventral margin of the skull in the orbitotemporal region on the alisphenoid bone and lateral pterygoid process. It runs posterodorsally to insert on the medial condyloid process, just below the condyle (Figs. 3, 8, and 9).

DISCUSSION The masticatory musculature of rodents is particularly unusual compared with that of other mammalian groups, especially with regard to the morphology of the masseter complex. It has been the subject of numerous studies over the years, and yet there still exist many dis- agreements and inconsistencies between these works, not only in nomenclature but also in anatomical detail as well. In particular, the description of the musculature of the rat by Hiiemae and Houston (1971), in which only two layers of the masseter are recognized (the zygomaticomandibularis being indistinguishable from the deep masseter in their view), is at odds with most other morphological work on rats (e.g. Turnbull, 1970; Weijs, 1973), and indeed other rodents (e.g., Wood and White, 1950; Wood, 1965; Woods, 1972; Gorniak, 1977; Ball and Roth, 1995; Thorington and Darrow, 1996; Druzinsky, 2010a). Yet, Hiiemae and Houston’s (1971) work has still been used as the basis for muscle anatomy in several other more recent articles (e.g., Byrd, 1981; Satoh, 1997, 1998, 1999; Bresin et al., 2000). The current study has highlighted these differences by presenting a review of previous work alongside morphological descriptions of the masticatory muscles of the squirrel, rat, and guinea pig, based on the new imaging technique of contrast- enhanced microCT. Three distinct layers of the masseter (superficial masseter, deep masseter, and zygomaticomandibularis) have been demonstrated in all three rodents under study (contra Hiiemae and Houston, 1971). The deep masseter has been shown to divide clearly into anterior and posterior parts in the squirrel and rat, but it is a much smaller muscle in the guinea pig and has not been separated into two portions. Druzinsky (2010b) found that the enlargement of the anterior deep masseter (i.e., sciuromorphy) increases the efficiency of biting at the incisors, so the reduced deep masseter of the guinea pig may indicate that it processes most of its food by chewing at the cheek teeth and spends less time gnawing at Fig. 11. Left lateral view of 3D reconstructions of the skull, mandi- the incisors than the squirrel or rat. The diets of the ble and masticatory muscles of (A) guinea pig, (B) rat and (C) squirrel rodents seems to support this inference, with rats and with the temporalis, superficial masseter and deep masseter removed. particularly squirrels consuming a great deal more hard azm, anterior zygomaticomandibularis; iozm, infraorbital part of zygo- food (nuts and seeds) than guinea pigs (Nowak, 1999). maticomandibularis; pzm, posterior zygomaticomandibularis. All scale However, it should be noted that Cavia porcellus has a bars: 5 mm. relatively larger superficial masseter and zygomaticomandibularis. Hence, the masseter forms a similar over- pterygoid runs ventrolaterally and fans out to make a all contribution ( 70%) to the masticatory musculature wide insertion on the medial surface of the angular pro- in the guinea pig and squirrel (see Table 1). No evidence cess, dorsal to the reflected insertion of the superficial for the posterior masseter (Woods and Howlands, 1979; masseter (Figs. 3, 8, and 9). Druzinsky, 2010a) was found in any of the three rodents. This is perhaps not surprising as it is not described in any dissections of Sciurus or Rattus (Turnbull, 1970; Weijs, 1973; Ball and Roth, 1995), and Woods (1972) External Pterygoid states that Cavia shows the least degree development of This muscle, like the internal pterygoid, varies little this muscle division among the hystricomorphs he in its morphology between the three rodents. However, studied. it is a much smaller muscle compared with its internal The temporalis muscle was shown to be considerably counterpart, forming just 3%–4% of the jaw-closing mus- larger in the rat than in the squirrel and the guinea pig. 926 COX AND JEFFERY

Fig. 12. Transverse lCT slices through head of (A) squirrel, (B) rat and (C) guinea pig. adm, anterior deep masseter; iozm, infraorbital part of zygomaticomandibularis; lt, lateral temporalis; mt, medial temporalis; t, temporalis. All scale bars: 5 mm. Slice positions shown by dashed lines.

Hiiemae (1971) suggests that the anterior temporalis tication is still relatively small when compared with functions most effectively at the power stroke of mastica- other hystricognaths, particularly those in the Octodon- tion, so the enlarged temporalis of the rat may indicate toidea (Vassallo and Verzi, 2001). This is largely due to a powerful molar bite in this species. The temporalis the mandibular morphology in the Caviidae, specifically was split into two sections (lateral and medial) in the a low mandibular condyle and a distally positioned rat and squirrel, but such a division was not clear in the angular process. guinea pig. It has been suggested that the two parts of We confirm that staining of biological specimens with the temporalis operate independently, the lateral (or an- iodine solution has enabled visualization of both the terior) part serving to stabilize and elevate the mandible hard and soft tissues of the skull using microCT imaging (Hiiemae, 1967). So, in rodents in which the mode of (Metscher, 2009; Jeffery et al., 2011). In this study, the mastication is mostly propalinal grinding, as opposed to contrast-enhanced microCT technique has been particu- crushing, the lateral temporalis is reduced (Klingener, larly useful for examining the zygomaticomandibularis 1964; Woods, 1972). Hence, the absence (or extreme muscle. Because of its origin on the medial surface of reduction) of the lateral temporalis in the guinea pig the zygomatic arch, it is very difficult to dissect out—the could be an adaptation to a lifestyle in which the action superficial and deep masseters have to be removed and of the molars is almost always grinding and hardly ever part of the zygomatic arch has to be cut away. However, crushing. In contrast, in squirrels and rats, where prop- the zygomaticomandibularis and its spatial relationship alinal chewing is much less important, the lateral tem- with the other masticatory muscles, the skull, and lower poralis is present, although its origin on the fascia jaw can be seen very clearly with the aid of contrast- overlying the medial temporalis is slightly more re- enhanced microCT (see Figs. 1–3, 8, 9). The enhanced stricted in Sciurus in this study than is indicated in any microCT also enables the creation of three-dimensional of the squirrel genera studied by Ball and Roth (1995) or reconstructions (Figs. 4, 7, and 11) and measurements Thorington and Darrow (1996). of, for example, muscle mass (see Table 1). It is felt that The internal pterygoid was shown to be particularly contrast-enhanced microCT will become an important large in the guinea pig, compared with the rat and tool in the study of soft tissue morphology and will be of squirrel. Contraction of this muscle produces lateral particular interest to researchers in the field of biome- movement (Hiiemae, 1971), which suggests that the chanical analysis, particularly finite element analysis guinea pig molar bite has a greater lateral component and multibody dynamic analysis, for whom accurate than that of the other two rodent species. Previous elec- knowledge of muscle geometry is essential. tromyographical studies indicate that this is indeed the case: although there is a lateral component to the rat ACKNOWLEDGMENTS molar bite, it appears to be small (Hiiemae and Ardran, 1968; Hiiemae, 1971), whereas Byrd (1981) noted a The authors thank Michael Fagan and Sue Taft, strong lateral component in the guinea pig masticatory Department of Engineering, University of Hull and Chris cycle. However, the lateral component of guinea pig mas- Martin, School of Materials, University of Manchester for RODENT MASTICATORY MUSCLES 927 assisting with the imaging. 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