Mediation of Muscular Control of Rhinarial Motility in Rats by the Nasal

Mediation of Muscular Control ofRhinarial Motility in Rats by the Nasal

Cartilaginous SkeletonSEBASTIAN HAIDARLIU,1* DAVID KLEINFELD,2 AND EHUD AHISSAR1

1Department of Neurobiology, the Weizmann Institute of Science, Rehovot, Israel2Department of Physics and Section of Neurobiology, University of California at San Diego,

La Jolla, California

ABSTRACTThe rhinarium is the rostral-most area of the snout that surrounds

the nostrils, and is hairless in most mammals. In rodents, it participatesin coordinated behaviors, active tactile sensing, and active olfactory sens-ing. In rats, the rhinarium is firmly connected to the nasal cartilages,and its motility is determined by movements of the rostral end of thenasal cartilaginous skeleton (NCS). Here, we demonstrate the nature ofdifferent cartilaginous regions that form the rhinarium and the nasofacialmuscles that deform these regions during movements of the NCS. Thesemuscles, together with the dorsal nasal cartilage that is described here,function as a rhinarial motor plant. Anat Rec, 296:1821–1832,2013. VC 2013 Wiley Periodicals, Inc.

Key words: nasofacilal muscles; rhinarium; rodents; vibrissa;whisking

INTRODUCTION

In rodents, rostral structures of the face are mobileand participate in active tactile and olfactory sensing.These structures include sensory hairs, such as macrovi-brissae and microvibrissae (Brecht et al., 1997), and therhinarium with its prominent narial pads (Ade, 1999;Hill, 1948) or nasal tubercles (Smith and Monie, 1969).The morphology and innervation of the rhinarium havebeen described in detail in several species, that is, coatiand raccoon (Barker and Welker, 1969), opossum (Pubolset al., 1973), cat (Abrahams et al., 1987), elephantshrews (Kratzing and Woodall, 1988), and moles (Cata-nia, 2005). Recent studies of vibrissa movements haveresulted in several biomechanical models of the vibrissamotor plant that relate muscle activation to the patternsof whisking (Berg and Kleinfeld, 2003; Knutsen et al.,2008; Haidarliu et al., 2011; Simony et al., 2010; Solo-mon and Hartmann, 2011). Yet the motile aspect of thestructure of the rhinarium remains to be characterized.

In rodents, rhinarium movements are involved in thefunctioning of two sensory modalities: active tactile sens-ing (Catania, 2005; Kleinfeld et al., 2006), and activeolfactory sensing (Haidarliu et al., 2013; Wachowiak,2011). Active tactile sensing is achieved by facilitation of

object touch by narial pads. These pads are denselyinnervated (Macintosh, 1975; Silverman et al., 1986),and are coated with glabrous skin that is topped by epi-dermal ridges, known as rhinoglyphics (Hill, 1948).Olfaction is based on air sampling by the nostrils duringsniffing (Kepecs et al., 2006; Wilson and Sullivan, 1999).Further, nose-to-nose touch may transmit behaviorally

Abbreviations used: DNC 5 dorsal nasal cartilage; ECM 5 extracellularmatrix; NCS 5 nasal cartilaginous skeleton.

Grant sponsor: The Minerva Foundation funded by the Fed-eral German Ministry for Education and Research; Grant spon-sor: The United States National Institutes of Health; Grantnumber: NS058668. Grant sponsor: The United States—IsraelBinational Science Foundation; Grant number: 2011432.

Ehud Ahissar holds the Helen Diller Family ProfessorialChair of Neurobiology.

*Correspondence to: Sebastian Haidarliu, Department of Neuro-biology, the Weizmann Institute of Science, Rehovot 76100, Israel.Fax: 972 8 9346099. E-mail: [email protected]

Received 26 June 2013; Accepted 29 August 2013.

DOI 10.1002/ar.22822Published online 29 October 2013 in Wiley Online Library(wileyonlinelibrary.com).

THE ANATOMICAL RECORD 296:1821–1832 (2013)

VVC 2013 WILEY PERIODICALS, INC.

important information that can alter subsequent socialinteractions in rats (Wolfe et al., 2011). Other mammalsthat possess rhinaria and associated musculature,appear to use them for more restricted behaviors. Coatiuse their rhinaria for tactile exploration (Barker andWelker, 1969), moles, for searching for food (Catania,2005), and Suoidea, for manipulations with force appli-cation (Herring, 1972). Lastly, in mammals, such as thehairy-nosed otter and wombat, short hairs cover the rhi-naria (Nguyen et al., 2001; Triggs, 1996), which suggestthat the rhinarium cannot mediate tactile sensing bydirect touch.

In the rat, the movable part of the nose is short,amplitudes of the movements are small, and the denselyinnervated surface of the rhinarium is restricted to twosmall narial pads. Nevertheless, these narial pads areused for tactile exploration of rostrally located objects.Rats turn their noses in different directions duringexploratory behavior and sniffing and, before takingdecisions, they touch the objects with their rhinarium(Bracha et al., 1990; Welker, 1964). Structural featuresthat can enable the rat nose, rhinarium, and narial padsto change their shape and touch objects appeared to beinterconnected. These include the nasofacial muscula-ture and the cartilaginous nasal capsule (Maier, 2000;Wang et al., 1994) also referred to as the nasal cartilagi-nous skeleton (NCS; Banke et al., 2002; Mess, 1999).

Recently, we determined the origins and insertionsites of several nasofacial muscles to evaluate their par-ticipation in vibrissa movements and control of the spa-tial patterns of nasal air conducting pathways(Haidarliu et al., 2010, 2013). The role of these musclesin whisking and general airflow control has been dis-cussed (Deschenes et al., 2012; Moore et al., 2013). Herewe seek to understand how rats achieve highly motilemovements with their nose as well as movements com-mon to whisking and sniffing. We determine the typicalgeometry of the nasal musculature and configuration ofthe cartilaginous structures of the outer nose, as well astheir participation in spatial changes in the nose tipposition. Further, the relationships between the NCSand the bony skeleton are examined to determinewhether and how they contribute to active touch by rhi-narium and narial pads.

MATERIALS AND METHODS

The morphology of the rhinarium and the basis of itshigh mobility were examined by histoenzymatic and his-tochemical methods that reveal anatomical structures insitu, without affecting tissue morphology during process-ing. All the experimental procedures were conductedaccording to NIH standards, and were approved by theInstitute Animal Care and Use Committee at the Weiz-mann Institute of Science. Tissue samples were obtainedfrom six 1-week-old, four 2-week-old, four 3-week-old,ten 12—14n-week-old, three 9-month-old, and four 1-year-old male albino Wistar rats. After slicing the tissuesamples with a microtome, striated muscles were visual-ized in even numbered slices by histoenzymaticallyrevealing cytochrome oxidase activity as recentlydescribed (Haidarliu et al., 2010). Nasal cartilages wereidentified and their relationships with the other snoutstructures determined in odd numbered slices histo-chemically. Nose retraction/protraction and vibrissa posi-

tion in behaving rats was determined simultaneouslyusing a regular Olympus camera (FE-330). Rhinarialmorphology in head-fixed rats was visualized with a bin-ocular digital stereo microscope (Labomed DigiStar;Labo America, Port Myers, FL).

Histoenzymatic Staining for CytochromeOxidase

After euthanasia with Penthal (0.6 mL/kg bodyweight, intraperitoneal), transcardial perfusion of ratswas performed with 4% (wt/vol) paraformaldehyde and5% (wt/vol) sucrose in 100 mM phosphate buffer (pH7.4). After perfusion, the rostral part of the snout wasexcised and postfixed in the perfusion solution with 30%(wt/vol) sucrose. After 48 hr of postfixation, each tissuesample was sectioned into 30 mm thick slices in the coro-nal, tangential, or horizontal planes using a slidingmicrotome (SM 2000R; Leica Instruments, Germany)coupled to a freezing unit (K400; Micron International,Germany). Slices in which cytochrome oxidase activitywas to be revealed were treated according to our modifi-cation (Haidarliu and Ahissar, 2001) of a procedure byWong-Riley (1979). Briefly, free-floating slices were incu-bated in an oxygenated solution of 4% (wt/vol) sucrose,0.02% (wt/vol) cytochrome c (Sigma-Aldrich, St. Louis,MO), catalase (200 mg/mL), and 0.05% (wt/vol) diamino-benzidine in 100 mM phosphate buffer for 2–3 hr atroom temperature under constant agitation. Upon cleardifferentiation between highly reactive and nonreactivetissue structures, the incubation was arrested by rinsingwith 100 mM phosphate buffer. Stained slices weremounted on slides, cover-slipped with Krystalon (Har-leco; Lawrence, KS), and examined by light microscopy.Striated muscles appeared as dark-brown bundles in therostral part of the rat head.

Histochemical Staining for Cartilage

In mammals, the nasal septum and entire NCS arecomposed of hyaline cartilage (Naumann et al., 2002;Popko et al., 2007). The hyaline cartilage of the NCSwas stained with alcian blue, which stains extracellularmatrix (ECM) blue, reveals different zones of hyalinecartilage (Conklin, 1963), and can bind to proteoglycansproportionally to their concentration (Ippolito et al.,1983). Since tissue components, such as cytoplasm,nuclei, and fibers are not stained by alcian blue, wecounterstained with thiazine red to visualize these struc-tures. Thiazine red has a high affinity for fibrillarystructures (Uchihara et al., 2000), stains myosins andepithelial cells, and emits orange (550 nm) fluorescence(Bacskai et al., 2003; Puchtler et al., 1974).

Odd-numbered, 30 mm-thick slices, which remainedunstained after staining for cytochrome oxidase activity,were mounted on slides, dried in air, and then stainedfor 30 min with 0.2% (wt/vol) alcian blue 8GX (Sigma-Aldrich) at room temperature. The slices were washedwith distilled water, and counterstained with 1% (wt/vol)thiazine red for 1 min at room temperature. After which,the slices were washed with distilled water, dehydratedin sequential solutions of 50, 70, 95% (wt/vol) and abso-lute ethanol, cleared in xylene, and cover-slipped withKrystalon. Some sections were also stained with

1822 HAIDARLIU ET AL.

methylene blue, neutral red, or cresyl violet (Haidarliuand Ahissar, 1997).

Stained slices were examined using a Nikon Eclipse50i microscope. Bright-field and/or epifluorescenceimages were imported into Adobe Photoshop software.Only minimal adjustments in the contrast and bright-ness of the images were made.

We examined whether the combination of alcian bluewith thiazine red can be useful for differential stainingof different types of cartilages, depending on the domi-nating type of collagen. As a positive control for reveal-ing hyaline and fibrous cartilage, we used slices fromthe knee of young rats that contain articular hyalinecartilage rich in Type II collagen, and ligaments, such asknee cruciate ligament, and Achilles tendon related tothe talocrural joint, which are reported to contain pre-dominantly Type I collagen (Eyre and Wu, 1983). Thearticular hyaline cartilage was extensively stained withalcian blue. Counterstaining with thiazine red did notchange the blue appearance of the hyaline cartilage,whereas other structures, which contained mostly Type Icollagen, appeared red. When the fluorescence of thecounterstained slices was examined, the hyaline carti-lage did not fluoresce, whereas structures containingType I collagen as a dominant component, that is, cruci-ate ligament, patellar tendon, Achilles tendon, and epi-dermis, exhibited strong red-yellow fluorescence.

Anatomical Description

A uniform description of nasal cartilages and musclesis still lacking (Bruintjes et al., 1996), despite efforts tounify anatomical nomenclature. For the facial muscles,terminology conforming to the Terminologia Anatomica(1998), with corresponding English equivalents (Whit-more, 1999), was used. Unifying nomenclature for thefacial musculature of mammals (Diogo et al., 2009) wasalso considered. We used cartilage terminology summar-ized and proposed by Zeller et al. (1993), Ade (1999), andMess (1999). Most cartilages in the external nasal skele-ton, cranial terminology in Chiroptera proposed byG€obbel (2000), and the description of rostral nasal carti-

lages in soricids (Maier, 2002) appeared relevant for therat as well. The external rat rhinarium morphology wassimilar to that of the lagomorpha described by Ade(1999).

RESULTS

Structural Organization of the Rhinarium andNCS

In the rat, the rhinarium is positioned at the rostral-most end of its snout. It involves the nostrils and twosymmetric narial pads recently described (Haidarliuet al., 2013). Each narial pad has two asymmetric parts,the crus superius and the crus inferius, similar to thosein rabbits (Ade, 1999). The dorsal and ventral borders ofthe rat’s narial pads are defined by two slender horizontalintegumental hairless folds, the lateral borders form themedial edge of the nostrils, and the medial borders aredetermined by the median sulcus. The surface of the crussuperius is covered by semicircular epidermal ridges (Fig.1). The crus inferius is smaller, and only slightly ridged,mainly at the border with the crus superius. These ridgescontinue up to the lateral edge of the narial pads.

In the resting state, the tip of the rat nose usuallyprotrudes rostrally beyond the line formed by the outermargin of the upper lip. When the vibrissae protract,the tip of the nose retracts (Welker, 1964). We wonderedif the nose retraction relative to the entire head was realor only apparent, particularly since the upper lip andmystacial pads also can be moved rostrally and result invibrissa movement described as combined vibrissa andmystacial pad translation (Mehta et al., 2007; Knutsenet al., 2008; Knutsen and Ahissar, 2009; O’Connor et al.,2010; Solomon and Hartmann, 2011; Moore et al., 2013).Turning of the head or unilateral vibrissa–object interac-tion during whisking leads to whisking and snout asym-metry (Ahissar and Knutsen, 2008; Deutsch et al., 2012;Grant et al., 2012; Mitchinson et al., 2011). Therefore,we dissected the rat rostrum such that the connectionsof the nasal capsule with the skull were preserved. Dis-sected NCSs from 1–2-week-old and 1-year-old rats

Fig. 1. Light microscopy of the entire rhinarium (a) and left narial pad (b) of an adult rat. (1) Dorsal integ-umental fold; (2) crus superius of the narial pad; (3) horizontal groove; (4) external nasal opening; (5) crusinferius of the narial pad; (6) ventral integumental fold; (7) median sulcus; and (8) epidermal ridges. M,medial; V, ventral. Scale bars 5 1 mm.

RHINARIAL MOTOR PLANT 1823

looked similar, except for their size (Fig. 2). The prepara-tions had cylindric shape, with a few prominent struc-tures, such as nasal tectum, dorsal nasal cartilage(DNC), and two symmetric cupular cartilages, whichwere giving a characteristic external appearance of thenasal capsule. When weak manual pressure was appliedto the tip of the nose in preparations obtained fromadult rats, the rostral end of the nose could be moved inthe caudal, dorsal, or ventral directions by �1 mm, andlaterally by >2 mm. When the pressure ceased, the tipof the nose returned back to its initial resting position.

Relationships of the NCS With the Rhinariumand the Skull

Examination of the rhinarium and adjacent structuresin histochemical preparations revealed the anatomical fea-tures that can account for described nasal mobility. First,the rhinarium and narial pads moved together with therostral end of the nose. Second, the narial pads weretightly attached to the cartilaginous lateral ventral process(Fig. 3) and could be separated from the cartilage only by

cutting the fibrous tissue that connects them. In contrast,the furry skin covering the remainder of the nose was eas-ily separated from the surface of the cartilage.

All nasal cartilages were assembled within the nasalcapsule into a unique cartilaginous ensemble that wasseen in both horizontal (Fig. 3) and coronal (Fig. 4) sli-ces. At the level of the pyriform aperture, the NCSformed a telescopic connection with nasal bones and pre-maxilla (Figs. 2, 3, and 5), which permitted a shift of theNCS relative to the skull along its rostrocaudal axis,and bending of the nose in the dorsoventral and lateraldirections. In the rat skull preparation, the rostral endsof the premaxilla tilted laterally, forming an openingreminiscent of a funnel, which may facilitate turningentire NCS in different directions (Fig. 6). Rostrolateraledges of the nasal bones formed an angle with the pre-maxilla, with the vertex located approximately 2 mmmore caudal than the rostral ends of the nasal bonesand of the dorsorostral spine of the premaxilla. Suchbone connections permit a larger lateral bending of theNCS relative to its bending in the dorsal and ventraldirections.

Fig. 2. Dorsal views of the nasal cartilaginous skeleton in a 2-week-old (a) and a 1-year-old (b) rats. (1)Cupular cartilage; (2) dorsal nasal cartilage attached to the nasal bones (3); (4) premaxilla; (5) externalnasal opening; (6) lateral ventral process; (7) nasal sidewall; and (8) nasal tectum. Scale bars 5 1 mm.

Fig. 3. Light microscopy (a) and fluorescence (b) of a horizontal sliceof the nasal cartilaginous skeleton of a 1-week-old rat after stainingwith alcian blue and counterstaining with thiazine red. (1) Narial pads;(2) lateral ventral process; (3) atrioturbinate; (4) septum; (5) septal

fenestra; (6) premaxilla; (7) maxilloturbinate; (8) ostium of the nasolacri-mal canal; (9) nasofacial muscles attached to the nasal sidewall; and(10) nasal cartilaginous skeleton. Scale bars 5 1 mm.


Intrinsic Organization of the NCS

The shape of the nose is maintained by the cartilagesof the NCS that are connected to each other directly orby means of intervening connective tissue and fibrousaponeurosis. Within the NCS, septum nasi was posi-

tioned in the sagittal plane. It was represented by ablade-shaped hyaline cartilage (Figs. 3 and 5) that hadan even thickness along the dorsal (Fig. 7a) and ventral(Fig. 7d) borders, but contained thin areas lacking carti-lage, that is, septal fenestrae, in the rostral half at the

Fig. 4. Light microscopy and fluorescence of nasal cartilages of anadult rat. (a) Coronal slice of the entire nasal cartilaginous skeletonstained with alcian blue and counterstained with thiazine red. (b)Boxed area in (a). Boxed areas in (b) represent roof cartilage (c) andthe ventral area/compartment of the dorsal nasal cartilage (d). (e andf) Blue autofluorescence and thiazine red fluorescence, respectively, of

the area shown in (b). (1) Septum; (2) roof cartilage; (3) dorsal nasalcartilage; (4) dorsal and (5) ventral compartments of the dorsal nasalcartilage; (6) chondrocytes clustered in groups; (7) matrix; and (8) peri-chondrium. Scale bars are 1 mm (a), 0.5 mm (b, e, and f), and 0.1 mm(c and d).


level of nostrils and narial pads (Figs. 3, 5a, and 7b,c)that facilitated NCS bending and nose retraction.

The NCS was represented rostrally by cupular carti-lage and its lateral ventral process, and continued cau-dally, along the midline, as the septal cartilage. Dorsally,

in caudal direction, cupular cartilages continued as roofcartilages to form, according to Maier (2002) the nasaltectum (Figs. 2 and 4). Laterally, the tectum was con-nected to the cartilages of the nasal sidewall. At thislevel, �3 mm caudal from the rhinarium in adult rats,the cartilage that formed the bottom of the nasal cavity(anterior transverse lamina), was fused with the septalcartilage, and connected to the cartilages of the nasalsidewall, which together with the tectum formed a con-tinuous cartilaginous ring (Fig. 5b4). A similar completecartilaginous ring, formed by nasal cartilages, was previ-ously observed in Echidna aculeata (Gaupp, 1908) andnamed Zona annularis. This ring was also described inguinea pigs (Dierbach, 1985), mouse lemurs (Lozanoffand Diewert, 1989), and in fetal tamarins (Smith et al.,2008) and tenrecs (Schunke and Zeller, 2010). In othermammals, such as the red squirrel (Slab�y, 1991), galago(Kanagasuntheram and Kanan, 1964), and adult tamar-ins (Mueller, 1981), the Zona annularis appeared to beinterrupted.

Changes in the configuration of the lateral nasal walloccur mainly in the portion of the NCS that occupies asegment between the nostrils and Zona annularis. Alongthis segment, cartilages of the nasal sidewall were notconnected to both the tectum in the dorsal part, and tothe anterior transverse lamina in the ventral part (Fig.6b3). When muscles originating from the nasal sidewallcontract, the nasal wall can be extended laterally inde-pendently from the rest of the NCS. Such an extensionis facilitated by the pliability and resilience of the hya-line cartilage.

Dorsal Nasal Cartilage

In dissected preparations (Fig. 2), the NCS slips cau-dally under the DNC, which overhangs the pyriformaperture from the dorsal, and forms a rostral continua-tion for the nasal bones. This nasal cartilage covers the

Fig. 5. Light microscopy of horizontal and coronal slices of the ratsnout stained for cytochrome oxidase activity. (a) A horizontal slice cutthrough the snout of a 17-day-old rat at the ventral margin of the nos-tril. (b) Light microscopy of the coronal slices cut at the positionsshown at the bottom of the panel (a). (1) atrioturbinate; (2) rostral edge

of the atrioturbinate; (3) narial pads; (4) ostium of the nasolacrimalduct; (5) lateral ventral process; (6) septal Fenestrae; (9) fragments ofthe intraturbinate muscles; (10) the bone of the maxilloturbinate; (11)rostral edge of the premaxilla; and (12) caudal part of the nasal carti-laginous skeleton. Scale bars 5 1 mm.

Fig. 6. Light microscopy of the rostral walls of the pyriform aperturein an adult rat skull preparation. (1) Nasal bones; (2) vertex of the angleformed by rostral edges of the nasal bones and premaxilla; (3) the slope(funnel) formed by the rostrolateral edge of the premaxilla; (4) incisives;(5) dorsorostral spine of the premaxilla. Scale bar 5 1 mm.


caudal half of the dorsal surface of the NCS in a visor-like fashion (DNC in Figs. 2 and 4).

The DNC may serve a second function. Besides limitingdorsal bending of the NCS, the DNC returns the NCS tothe resting position after deflection. It may also play aprotective role for such a flexible structure as the NCS,preventing its dorsal surface from mechanical impacts.This cartilage has specific tinctorial properties and fluo-rescence characteristics. The DNC is composed of twosymmetric parts that are fused along the midline. In theventral half of each part, there is a horizontally positionedand compressed in dorsoventral direction structure con-

sisting of hyaline cartilage (Fig. 4a,b,d), which was notstained with thiazine red and remained blue color. Thedorsal compartment of each part was extensively stainedwith thiazine red. These dorsal and ventral compartmentswere surrounded by a perichondrium that was also stain-able with thiazine red (Fig. 4a,b). Thiazine red stainingwas detected by light microscopy, and by red fluorescence(Fig. 4f). The blue autofluorescence of the septal cartilageand of both the dorsal and ventral compartments of theDNC was similar (Fig. 4e). Such autofluorescence is gen-erally characteristic for different types of collagen, majorcomponents of cartilages (Kutsuna et al., 2010).

Fig. 7. Light microscopy of stained for cytochrome oxidase activityhorizontal slices of the rostral part of the 1-week-old rat snout in thedorsal part of the nasal cavity (a), at the level of nostrils and narialpads (b and c, respectively), and at the level of anterior transverselamina (d). (1) Septum; (2) aponeurosis above Cupula nasi; (3) M. dila-

tor nasi; (4) olfactory bulb; (5) ethmoturbinates; (6) Fenestrae septinasi; (7) incisives; (8) dorsal edge of the nostril; (9) atrioturbinate; (10)ventral edge of the nostril; (11) sites of muscle attachments to thenasal sidewall; and (12) anterior transverse lamina. Scale bars 5 1mm.

Fig. 8. Muscle attachments to the cartilages of the nasal sidewall. (a and b) Light microscopy and (c)blue autofluorescence; (b) and (c) represent boxed area in (a). (1) Rhinarium; (2) atrioturbinate; (3–5) super-ficial, pseudointrinsic and posterior slips of the Pars interna, respectively; and (6) Pars anterior of the M.nasolabialis profundus. RM, rostromedial; V, ventral. Scale bars 5 1 mm.


Musculature of the NCS

Most parts of the M. nasolabialis profundus, that is,the Partes maxillares superficialis et profunda, Parsinterna profunda, superficial, pseudointrinsic, and pos-

terior slips of the Pars interna, originate from thenasal sidewall (Figs. 3, 5a,b3, and 8), so their unilat-eral contraction can move the rostral part of the NCSlaterally, change the angle between the axes of theNCS and of the head, translate rostrally correspondingmystacial pad, and modify the pattern of the atriumconfiguration. Bilateral contraction of these musclescan move entire NCS caudally due to telescopic connec-tion of the NCS with the skull, and translate both mys-tacial pads rostral due to insertion of these musclesinto mystacial pads. The tendon of the M. dilator nasiinserts into the aponeurosis above the NCS (Fig. 7a),and can move the rostral part of the NCS and rhina-rium in the lateral and/or dorsal directions (Haidarliuet al., 2013).

The rostral set of the superficial slips of the Parsinterna (Figs. 5b2 and 9a) may have a special role in rhi-narial movements. These muscle bundles originate fromthe nasal sidewall and ventral surface of the atrioturbi-nate, rostral to the ostium of the nasolacrimal duct.They are directed dorsally, encompassing the Cupulanasi from the lateral and dorsal, and are inserted intothe collagenous layer of the skin close to midline, abovethe rhinarium (Fig. 9b). These muscle slips may retractthe skin above the Cupula nasi, rotate the rhinariumslightly, and simultaneously raise the base of the atrio-turbinate, which may change the pattern of the air con-ducting pathways in the atrium.

Based on anatomical data about the origins and inser-tion sites of the rostral-most nasofacial muscles, and onspatial relationships between the muscles, NCS, and rhi-narium, a "rhinarial motor plant" consisting of theseanatomical structures is proposed. The muscles in thismotor plant include the M. levator rhinarii, M. depressorrhinarii, M. depressor septi nasi, M. dilator nasi, rostralslips of the M. transversus nasi, and six parts/slips thatbelong to the M. nasolabialis profundus, that is, Partesmaxillares superficialis et profunda, Pars interna pro-funda, and pseudointrinsic, superficial, and posteriorslips of the Pars interna. The position of all thesemuscles is schematically shown in Fig. 10.

Fig. 9. Light microscopy of a coronal (a) and a tangential (b) slices of the rostral part of the rat snoutstained for cytochrome oxidase activity. (1) Superficial slips of the Pars interna of the M. nasolabialis pro-fundus; (2) Cupula nasi; (3) atrioturbinate; (4) rhinarium; and (5) nasal sidewall. A4 and B4, vibrissal fol-licles; RM, rostromedial; V, ventral. Scale bars 5 1 mm.

Fig. 10. Schematic image of muscles serving rhinarial motor plant.(1–6) Partes maxillares superficialis et profunda, Pars interna profunda,and posterior, pseudointrinsic, and superficial slips, respectively, of thePars interna of the M. nasolabialis profundus; (7) rostral slips of the M.transversus nasi; (8) tendon of the M. dilator nasi; (9) M. levator rhinarii;(10) M. depressor septi nasi; and (11) M. depressor rhinarii. Encirclednumbers indicate muscles not shared with other motor plants.


DISCUSSION

Involvement of Task-Aimed Nasofacial MuscleClusters in Rhinarium Movement

We confirmed that only two nasofacial muscles origi-nate in the rhinarium (Mm. levator et depressor rhi-narii). However, contraction of these two muscles cannotaccount for all rhinarial movements. These two smallmuscles can stretch moderately integumental folds inthe vertical direction according to their origin and inser-tion sites, and simultaneously stabilize the position ofnarial pads during object touch, and probably only movethe rhinarium up or down slightly. The amplitude of rhi-narium movements depends on their directions. Verticalmovements are limited by two anatomical structures: inthe ventral direction, by the dorsorostral spine of thepremaxilla, and in the dorsal direction, by the DNC thatis connected to the nasal bones and covers the caudalhalf of the NCS. Horizontal (lateral) rhinarial move-ments, which do not have such limits, have larger ampli-tudes. These horizontal movements of the rhinarium canbe called translational because they are determinedentirely by the deflections of the rostral end of the NCS.Furthermore, these movements may be synchronizedwith the rostral translation of mystacial pads due to con-traction of different parts and slips of the M. nasolabia-lis profundus, because these muscles originate from thenasal sidewall (posterior and pseudointrinsic slips of thePars interna, Pars interna profunda, and Partes maxil-lares superficialis et profunda), and are inserted into themystacial pad. The participation of these muscles inboth sniffing (fast breathing) and whisking adds a poten-tial anatomical coupling link to the circuitry that drivesbreathing (Smith et al., 1991) and whisking (Mooreet al., 2013).

The organization of the nasofacial musculature intodifferent motor plants according to their spatial arrange-ment and sensory tasks can be summarized (Table 1)based on data by Hill et al. (2008) about the vibrissamotor plant, and data about arrangement of the musclesin the mystacial pad (Haidarliu et al., 2010) and theirparticipation in active olfactory sensing during sniffing(Haidarliu et al., 2013). The rhinarial motor plant isserved by 11 muscles or muscle slips, of which seven areshared with both vibrissal and nasal olfactomotor plants,two are shared only with the olfactomotor plant, andtwo are only involved in rhinarial movements (M. leva-tor and M. depressor rhinarii). The vibrissal motor plantis served by all intrinsic and 12 extrinsic (for mystacialpad) muscles, whereas the nasal olfactomotor plant isserved by 11 nasofacial muscles. The nasal olfactomotorand vibrissa motor plants share seven muscles.

Since most nasofacial muscles that belong to differentmotor plants can perform different tasks during theircontraction can explain why rhinarium, from which onlytwo muscles originate, can move in different directionsin three-dimensional space. This enables the rhinariumto perform an active tactile sensing by touching objects,and to be involved in other coordinated movements, suchas sniffing and airflow control.

Recently, few rat models aimed to accelerate recoveryafter facial nerve injury were developed (Angelov et al.,2007; Guntinas-Lichius et al., 2007; Hadlock et al., 2010;Heaton et al., 2013). In these models, mystacial pad waschosen as a target for mechanical stimulation. We sug-

gest that NCS also may serve as a similar target. Wefound that rhinarial motor plant possesses muscles thatoriginate from the NCS and insert into mystacial pads.Presumably, movements of the NCS force to move mys-tacial pads as well. To move NCS, mechanical stimula-tion or inhalation of chemical agents that provokesniffing can be used. Sniffing is accompanied by simulta-neous oppositely phased repetitive retraction and pro-traction of the tip of the nose and of the mystacialvibrissae (Welker, 1964). Due to reciprocating rostrocau-dal movements of the NCS during sniffing, mystacialpads can move synchronously. Consistent controlledsniffing/whisking can be evoked, for example, by inhala-tion of peanut butter or isopropyl alcohol (Heaton et al.,2008).

Nasal Cartilages as Mediators of RhinarialMovements

In rodents, the nose participates in active tactile andolfactory sensing, functions reflected in the nose anat-omy. Nasal and rhinarial mobility, which is functionallyimportant, is controlled by the movement of the rostralend of the NCS. The NCS was initially described in bats(Grosser, 1902), and later in many other mammals,including shrews (Kratzing and Woodall, 1988), rabbits(Mess, 1999), and mole rats (Banke et al., 2002). In rab-bits, cartilages of the NCS are hyaline cartilages, whichhave the highest stiffness and unique pattern of collagencomposition (Naumann et al., 2002). We suggest that theNCS determines nasal shape, rhinarial movements,narial pad active touch, and the pattern of air conduct-ing pathways within the vestibulum nasi in the same

TABLE 1. Nasofacial muscles involved in variousnasofacial motor plants

Nasofacial muscles

Rhinarialmotorplant

Nasalolfactomotor

plant

Vibrissamotorplant

M. levator rhinarii 1 2 2M. depressor rhinarii 1 2 2M. depressor septi nasi 1 1 2Intraturbinate muscle 2 1 2M. nasolabialis

profundus,Pars anterior

2 1 2

Pars interna, superficialslips

1 1 2

Pars interna profunda 1 1 1Pars interna,

posterior slips1 1 1

Pars interna,pseudointrinsic slips

1 1 1

Pars maxillarissuperficialis

1 1 1

Pars maxillaris profunda 1 1 1Pars media superior 2 2 1Pars media inferior 2 2 1M. dilator nasi 1 1 1M. transversus nasi 1 1 1M. buccinatorius, Pars

orbicularis oris2 2 1

M. maxillolabialis 2 2 1M. nasolabialis 2 2 1Intrinsic muscles of the

mystacial pad2 2 1


manner as colllagenous skeleton of the rat mystacial paddetermines the anatomical and functional integrity ofthe entire mystacial pad.

The morphology of the DNC in rats is described herefor the first time. Whereas most cartilages in the bodyare gradually replaced by bone, the rostral part of thenasal septum, which is centrally located in the NCS,remains cartilaginous throughout the life of mammals(Scott, 1953). Similarly, here we observed that in agedrats cartilages in the entire NCS and the DNC overlyingit retained their flexibility and had a similar appearanceand proportions as in young rats (Fig. 2), without signsof ossification. The structural organization of the DNCreflects its functional properties. The caudal end of thiscartilage is connected to the nasal bones. Although thiscartilage has no direct connections with the muscles, itis constantly subjected to mechanical action by the dor-sal surface of the NCS when the latter is moving orbending. The dorsal movement of the NCS may bedamped and limited by the DNC. These two structuresare linked together by connective tissue that forms anasal aponeurosis. When the muscles that moved theNCS in whatever direction become relaxed, DNC facili-tates the return of the NCS to the resting position. Thisoccurs because the core of the DNC contains resilienthyaline cartilage that works like a spring and damper,and is surrounded by a layer of fibrous cartilage withstretching and shock-absorbing properties. We suggestthat other mammals also may possess similar comple-mentary cartilaginous structures which can protect NCSfrom mechanical impacts and/or regulate using their rhi-narium for active tactile sensing by the nose.

The different tinctorial properties of the cartilage ofthe DNC compared to that of the NCS (Fig. 4) mayaccount for the DNC not being observed in previousstudies. When slices were stained with alcian blue andthen counterstained with other dyes, such as neutralred, methylene blue, cresyl violet, or thiazine red, onlythiazine red did not stain the NCS. With thiazine red,the blue color of the alcianophilic hyaline cartilageremained unchanged, whereas other structures that con-tained mainly Type I collagen were extensively stained(Figs. 3 and 4). Cresyl violet and neutral red, like alcianblue, have a high affinity for hyaline cartilage. Theyextensively stain only hyaline cartilage in the NCS andDNC, and not the dorsal area of the DNC (Fig. 11). Cati-onic dyes, such as alcian blue and neutral red, were

reported to bind to structures that contain glycosamin-glycans (Sulzer and Holtzman, 1986).

Alcian blue selectively stained the ECM of all the car-tilages of the NCS, and only the ventral part of the coreof the DNC (Fig. 4a,b). This tinctorial dissimilarity ofthe NCS and DNC can be explained by the presence ofdifferent collagen types. Type I collagen is present infibrous (tendinous) tissues and fibrocartilage, whereasType II collagen is characteristic of the ECM of the hya-line cartilage (Filova et al., 2010; Mizoguchi et al.,1992), and accounts for about two-thirds of the dryweight of articular hyaline cartilage (Buckwalter andMankin, 1998; Eyre, 2002). Blue autofluorescence,caused by all the types of collagen, was similar for theNCS and for both parts of the DNC (Fig. 4e), whereasred fluorescence after staining with thiazine redoccurred only in the dorsal part and in the fibrous peri-chondrial envelope of the DNC, and was absent in theNCS and in the ventral part of the DNC (Fig. 4f). Thesefindings agree with those of Mizoguchi et al. (1992) whofound that Type II collagen is characteristic for the sep-tal cartilage, whereas Type I collagen is absent in theECM of septal cartilage.

Narial pads play a central role in active tactile sens-ing by the nose. The pads are attached to the rostral endof the NCS that is represented by an ensemble of carti-lages that exhibit certain mobility when a behaving ani-mal moves its nose and/or interacts with objects. Suchmobility and flexibility of the rostral part of the ratsnout is provided by a telescopic connection between theNCS and the nasal and premaxilla bones that form theentrance (pyriform aperture) for the NCS into the nasalcavity of the skull, and by the ability of the NCS to bendalong its rostrocaudal axis. Nasofacial muscles of the ratsnout can move the entire NCS, together with the rhina-rium, in the caudal direction. However, in the rat, therole of the telescopic anatomical connection between theNCS and the bony walls of the pyriform aperture is min-imal for rhinarium retraction, being limited to �1 mmbecause of the strong fasciae that connect the dorsal andventral parts of the NCS to the bones. Dorsoventraldeflections of the rostral end of the NCS are also ofsmall amplitudes because dorsally, the DNC extends ros-trally from the nasal bones (Fig. 2), and ventrally, theprominent dorsorostral spine of the premaxilla forms themost rostral point of the skull (Fig. 6) and limits ventraldeflection of the NCS. In shrews, retraction of the nose

Fig. 11. Light microscopy of adult rat coronal snout slices stained with alcian blue (a), cresyl violet (b),and neutral red (c). (1) Septum; (2) maxilloturbinate; (3) roof cartilage; and (4 and 5) hyaline and fibrousareas, respectively, of the dorsal nasal cartilage. Scale bars 5 1 mm.


is due to telescopic arrangement of the three caudal-most elements of the NCS (Maier, 2002). In other ani-mals, such as Suidae, in which the rhinarium movesand can apply force to objects, movements are due tospecific anatomy and special musculature (Herring,1972).

Telescopic principle of connecting cartilage to the bonethat permits to move NCS relative to the skull along itssagittal axis, as well as the ability to produce angulardeflections of the nasal capsule may be appropriate forthe other animals. Further study of such connectionsmay be useful for understanding mechanisms of nasalmotility during active tactile sensing with the nose, aswell as for nasal surgery which can use this principle innose reconstruction after nasal injuries.

Summary

The rostral nasofacial muscles of the rat are function-ally organized into task-intended clusters that assist inwhisking, sniffing, and rhinarial movements. These canbe classified as vibrissa motor, nasal olfactomotor, andrhinarial motor plants, respectively (Table 1). Thesemotor plants are overlapping: each of them involvesmuscles that are shared with the other motor plants, aswell as muscles that perform only one specific task. Therhinarium is tightly attached to the rostral end of theNCS, the motility of which determines rhinarial move-ments. Movement of the NCS is controlled by rhinarialmotor plant, the muscles of which are partially sharedby both the vibrissa motor, and nasal olfactomotorplants. Muscles that change the atrioturbinate positionand shape, and muscles that dilate nasal airways, repre-sent the nasal olfactomotor plant that regulates sniffingparameters by shaping nasal air-conducting pathways.

ACKNOWLEDGEMENTS

The authors thank Barbara Schick for reviewing themanuscript.

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Mediation of Muscular Control of Rhinarial Motility in Rats by the Nasal