Indexing terms: cholera toxin; ear, external; horseradish peroxidase; sound localization

THE JOURNAL OF COMPARATIVE NEUROLOGY 363:600-614 (1995)

LUIS C. POPULIN AND TOM C.T. YINNeuroscience Training Program and Department of Neurophysiology,

University of Wisconsin, Madison, Wisconsin 53706

branches of the facial nerve and the subdivisions of thenucleus. With the exception of an abstract on the innerva­tion to pinna muscles of the cat (Zook et al., 1981); theorganization of motoneuron pools innervating any particu­lar facial muscle group has not been characterized in thisspecies. Kume et al. (1978) attempted to inject free HRPinto individual facial muscles in the cat, but difficulties dueto spread of the labeling enzyme into neighboring tissueforced them to inject groups of muscles instead. Thus thelocation of motoneurons that innervate the muscles of thepinna of the cat has been mapped only to the resolutionprovided by individual branches of the facial nerve (Kumeet al., 1978). This is not the case in other species. Detailedanatomical studies comparing the topographical organiza­tion of pinna motoneuron pools of the rat, which does notmove its pinnae very much, and the bat, which has verymobile pinnae, suggest that structural features such as thenumber of motoneurons and the degree of organizr-.tion ofthe motoneuron pools reflect the differences in pinnamobility between the two species (Friauf and Herbert,1985). If pinna mobility and anatomical organization arecorrelated, then we would expect the cat's mobile pinnae to

Accepted July 7,1995.Address reprint requests to Luis C. Populin, University of Wisconsin,

Department of Neurophysiology, 283 Medical Sciences Building, 1300University Avenue, Madison, WI 53706.

© 1995 WILEY-LISS, INC.

Topographical Organizationof the Motoneuron Pools That Innervate

the Muscles of the Pinna of the Cat

ABSTRACTThe topographical organization of the 22 motoneuron pools that innervate the pinna

muscles of the cat was examined by injecting the B-subunit of cholera toxin conjugated tohorseradish peroxidase into individual muscles. All 22 pools are found in the facial nucleus,organized as rostro-caudally oriented columns, and arranged according to the action of themuscles they innervate. Pools innervating muscles that pull the pinna dorsally are located inthe dorsal two thirds of the medio-dorsal subdivision, and those innervating muscles that pullthe pinna ventrally are located in the ventral one half of the nucleus. Motoneurons innervatingmuscles that pull the pinna cranially are located laterally, those that pull the pinna caudally arelocated medio-ventrally, and those that change the shape of the pinna are located along theentire dorso-ventral extent in the center of the medio-dorsal subdivision. This topographicallayout is consistent with the somatotopic organization of the entire facial nucleus asdemonstrated in a variety of species. Co? 1995 Wiley-Liss, Inc.

The pinna of the cat differentially filters acoustic signalsdepending upon the location of the sound source. Signalsdelivered on the acoustic axis, which is the position in thefree field where an acoustic signal produces maximal soundpressure level, are amplified and those delivered off theacoustic axis are attenuated (Wiener et al., 1966; Phillips etal., 1982; Calford and Pettigrew, 1984; Musicant et al.,1990; Rice et al., 1992). Unlike the pinnae of primates orhumans, which are relatively immobile, the pinnae of thecat move extensively, allowing the animal to selectivelyamplify or attenuate auditory signals from different loca­tions in space. Little is known about such movements andtheir functional significance, but roles in sound localization(Siegmund and Santibaiiez-H, 1981, 1982) and in theidentification of novel auditory stimuli (Henkel and Ed­wards, 1978) have been suggested.

There are 22 muscles associated with each pinna of thecat (Crouch, 1969). Sixteen of them participate directly orindirectly in pinna movements, and the other six change itsshape (Fig. 1). The motoneuron pools that innervate thesemuscles are found in the medial portions of the facialnucleus (FN; Papez, 1927; Courville, 1966; Henkel andEdwards, 1978; Kume et al., 1978; Vidal et al., 1988).

Prior anatomical studies of the FN of the cat, usingdegeneration techniques (Courville, 1966) and horseradishperoxidase (HRP) injections into muscle groups (Kume etal., 1978), examined the relationship between the different

INNERVATION OF PINNA MUSCLES OF THE CAT

be related with a high degree of organization among pinnamotoneuron pools in the FN.

In the present study we have characterized the topo­graphical organization of pinna motoneuron pools in theFN of the cat by injecting individual muscles with the Bsubunit of cholera toxin conjugated to HRP (CTB-HRP).The results show that the 22 pinna motoneuron pools areorganized in a manner consistent with the somatotopicorganization of the entire FN shown in other species. Poolsinnervating muscles with similar actions overlap to differ­ent degrees, but those innervating muscles with differentactions overlap very little or not at all. Preliminary resultshave been presented elsewhere (Populin and Yin, 1992).

MATERIALS AND METHODSAnimals and surgical procedures

Data from 17 cats (2.0-6.0 kg) are presented in thispaper. Anesthesia was induced with Ketamine/Aceproma­zine (20 mg/kg; 0.2 mg/kg, Lm.) and maintained duringsurgery with i.v. injections of sodium pentobarbital. Prepa­rations for surgery also included the administration of66,000 units/kg of penicillin and 0.022 mg/kg of atropinesulfate.

Horseradish peroxidase conjugated with CTB (List Labo­ratories) reconstituted with distilled water to a concentra­tion of 0.1% was injected into individual pinna muscles witha 10 fJ-1 microsyringe. Since early experiments showed that,with the exception of the M. intermedius scutulorum,labeling occurred in the ipsilateral FN only, two muscles,one on each side, were injected in most experiments. Tomaximize the number of muscle fibers reached by thelabeling enzyme, the CTB-HRP was delivered in a series ofsmall injections 0-2 fJ-I each), intended to cover the cross­section of the target muscle. The total volume ofCTB-HRPinjected into each muscle varied between 20-50 fJ-1 depend­ing upon the size of the muscle.

In most experiments the head of the cat was positioned ina stereotaxic frame, and the microsyringe was held with amanipulator. In other experiments the position and orienta­tion of the fibers of muscles such as those on the side of thehead (e.g., M. adductor auris medius, M. antitragicus, andM. adductor auris inferior) required us to hand-hold thesyringe. In general, to prevent wrinkling and tissue displace­ment, penetrations were made parallel rather than orthogo­nal to the orientation ofthe muscle fibers.

To minimize diffusion of the labeling enzyme into non­target tissue all muscles were exposed and isolated prior toinjection with the exception of the M. transversus auriculiand M. auricularis externus which were injected throughthe skin. The procedure normally included 1) identificationand expo ure of the target muscle, 2) removal or denerva­tion of neighboring tissue (part of the M. platysma wasremoved to expo e the M. depressor conchae and M.

RESULTSThe facial nucleus and CTB-HRP labeling

Figure 2 shows a series of coronal sections approximatelyevenly spaced through the FN and arranged from caudal torostral. In the central two thirds of the structure fourdistinct subdivisions are distinguished: 1) intermediate(INT), 2) lateral (LAT), 3) ventromedial (VM), and 4)mediodorsal (MD) (Fig. 2B-E). ear the caudal and rostralpoles the cross-sectional area of the nucleus is smaller, andthe INT, LAT, and VM subdivisions gradu.ally disappear(Fig. 2A,F).

601

Motoneuron pools:Topographical arrangement

We have classified the muscles of the pinna into twomajor functional groups: those that 1) move the pinnadirectly or indirectly and 2) those that change its shape. Wefurther subdivided the first group into four subgroupsaccording to their principal action: muscles that pull thepinna a) dorsally, b) ventrally, c) caudally, and d) cranially.Data concerning the action, origin, and insertion points ofeach muscle were taken directly from Crouch (1969). Inspecifying the actions of muscles that act in two directions,the primary action is given first. Thus a muscle which pullsthe pinna cranio-dorsally has a cranial action principally,whereas a muscle that pulls dorso-cranially has a dorsalaction principally.

Motoneuron pools innervating the muscles of the pinnaare found in the INT (two), the VM (one), and the MD

submentalis), and 3) sealing the area with silicon or vase­line. In addition we selected CTB-HRP over free HRPbecause it is a more efficient marker (Trojanowski et aI.,1982) that can be used at a much lower concentration (0.1%vs. 20-30%), which is thought to prevent diffusion (Weltand Abbs, 1990).

Histology and data analysisAfter a survival period of 48-96 hours, cats were given a

lethal dose of sodium pentobarbital intravenously 050 mg)and perfused intracardially with 1) saline and sodiumheparin, 2) LO~ paraformaldehyde and 2.5% glutaralde­hyde in 0.1 M phosphate buffer at pH 7.4 and 3) 591 sucrosein 0.1 M phosphate buffer pH 7.4. The brainstem wasremoved and stored in 3091 sucrose at 4°C for 24-48 hours.The portion of the brainstem containing the facial nucleuswas frozen and cut in 70 ILm sections in the coronal plane.All sections were processed with tetramethylbenzidine (Me­sulam, 1978) and counterstained with cresyl violet.

Results are presented in the form of camera lucidadrawings of coronal sections of the FN at approximately 25,50, and 75% of its length (measured from the caudal pole ofthe nucleus) in which labeled cells from two adjacentsections are plotted (see Figs. 5-9), and in the form ofphotomicrographs of coronal (see Figs. 2-4) and parasagit­tal (see Fig. 10) sections of the FN. The boundaries of theFN were drawn around the outer edge of the cell bodies inthe cresyl violet stained material. The different subdivi­sions of the FN were defined by areas without cell bodies.To facilitate the comparison between different muscles alldrawings are shown on the right side of the brainstem;drawings corresponding to experiments performed on theleft side are transposed.

ABBREVIATIONS

cholera toxin B horseradish peroxidasefacial nucleusintermediate subdivisionlateral subdivisionlateral superior olivemedio·dorsal subdivisionmedial superior oliveventro-medial subdivision

CTB·HRPFNINTLATLSOMDMSOVM

L.C. POPULIN AND T.C.T. YIN

Tragicus lateralis

Abductor AurisBrevis

cartilage and on the surface of the auricle; it pulls theexternal ear dorso-caudally. Injection of CTB-HRP in thismuscle produces a column of tightly packed labeled moto­neurons in the most medial areas of the MD subdivision oftheFN.

The M. auricularis superior runs from the sagittal crest,cranial to the interparietal bone, to the auricular cartilage.Its action is to adduct the external ear. The motoneuronpool innervating this muscle is more diffuse and locatedmore dorso-Iaterally than that innervating the M. levatorauris longus.

The M. adductor auris medius pulls the concha dorso­cranially. The origin of this muscle is in the middle twothirds of the caudo-ventral edge of the scutiform cartilage,and its insertion point is along the medial surface of thetragus. Its motoneurons are found in the dorsal region ofthe MD subdivision slightly lateral to its midline. Unlikethe motoneuron pools innervating the two precedingmuscles, these cells appear to be arranged in a sheet-likefashion.

The M. intermedius scutulorum runs between the scuti­form cartilage on both ears and pulls the two ears dorsally.CTB-HRP injection into one side of this muscle produceslabeled motoneurons in the dorso-Iateral aspects of the MD

Trag.cus Medialis

FrontoauricularisCorrugator Supercllii

lateralis

Abductor AurisLongus

levator Auris longus(caudal)

Fig. 1. Muscles of the pinna of the cat. Top view of the most superficial (upper lell), and deepermusculature (upper right). Side view with (lower left) and without (lower right) the platysma muscle (FromJ.E. Crouch, Text-Atlas of Cat Anatomy. Philadelphia, Lea and Febiger, 1969. Used with permission).

Adductor AUflsMedius

TransversusAuriculi

(nineteen) subdivisions, where they are topographicallyorganized; a few motoneurons innervating the M. zygomati­cus are also found in the most medial edge of the LATsubdivision. Pools that innervate muscles with similaractions tended to overlap while those that innervate muscleswith different actions tended to be spatially distinct (Fig. 3).In Figure 3 we contrast the location of motoneuronsinnervating two adjacent muscle that have different pri­mary actions: the M. adductor auris medius pulls the pinnadorsally while the M. adductor auris inferior pulls itcranially.

To give an indication of representative labeled cells,Figure 4 shows high-power views of labeled motoneuronsfollowing an injection of CTB-HRP into M. levator aurislongus. Labeled cells exhibited a variety of shapes andtended to be clustered together (Figs. 3, 4D).

Muscles that move the pinnaMuscles that pull the pinna dorsally. The motoneuron

pools that innervate this group of muscles are located in thedorsal two thirds of the MD subdivision of the FN (Fig. 5).

The M. levator auris longus (caudal subdivision) is a largemuscle that extends from the midline, dorsal to the atlas,and from the sagittal crest, and ends in the scutiform

602

603

four subdivisions of the nucleus are most clearly distinguished at thecenter (C): INT, intermediate, LAT, lateral, VM, ventro-medial, andMD, medio-dorsal. MSO, medial superior olive.

Fig. 2. Cresyl violet stained coronal sections of the right facialnucleus at approximately 10, 27, 50, 63, 80, and 93% of its length(measured from the caudal pole). A, caudal pole; F, rostral pole. The

INNERVATION OF PINNA MUSCLES OF THE CAT

604

Fig. 3. Coronal sections of the facial nucleus of the cat at approxi­mately 50% of its length illustrating the distinct location of twomotoneuron pools innervating muscles with different actions. A:

subdivision, in both ipsilateral and contralateral facialnuclei.

Muscles that pull the pinna ventrally. This group iscomposed of four muscles. Motoneuron pools innervatingthese muscles are located in the ventral half of the FN andin the lateral aspects of the MD subdivision (Fig. 6).

The M. zygomaticus major, which pulls the ear ventro­cranially, connects the angle of the mouth with the scuti­form cartilage. Consistent with the location of other pinnamuscles whose actions include a cranial component (seeFig. 8), the motoneuron pool ofthe M. zygomaticus major islocated in the lateral aspect of the MD subdivision, overlap­ping considerably with those pools innervating the M.frontoscutularis, the M. adductor auris superior, and theM. intermedius scutulorum. A few motoneurons are alsofound in the medial edge of the LAT subdivision, giving theimpression that the column winds from the MD to the LAT,and back to the MD subdivision as it runs rostro-caudally.

The M. submentalis, like the M. zygomaticus major, is along, wide and very thin ribbon of muscle fibers whichoriginates near the mid-ventral line and inserts into thepre-auricular aponeurosis. It pulls the external ear ven­trally. The column of labeled motoneurons is found in theventro-Iateral aspects ofthe MD subdivision.

The M. tragicus lateralis pulls the external ear ventrallyand rotates it outwardly. It originates on the caudal end ofthe mandible, and inserts on the caudal margin of thetragus and in the depression on the concha just caudal ofthe tragus. The motoneurons innervating this muscle arelocated in the ventral aspect of the MD subdivision.

The M. depressor conchae is also a long, wide, and verythin ribbon of fibers that originates on the ventral surfaceof the neck and inserts into the summit of the antitragus. Itpulls the external ear ventrally. This is the only pinnamuscle that is innervated by motoneurons located in theVM subdivision ofthe FN.

Muscles that pull the pinna caudally. This group iscomprised of three muscles, innervated by motoneuronslocated medio-ventrally in the MD subdivision of the F(Fig. 7).

The M. abductor auris brevis pulls the concha caudally. Itis a small, thin, and narrow ribbon of fibers located

L.C. POPULI AND T.e.T. YI

Motoneurons labeled after injection into the M. adductor auris medius.B: Motoneurons labeled after injection into the M. adductor aurisinferior.

immediately caudal to the M. abductor auris longus; it runsfrom the lambdoidal crest to the medial surface of theconcha. Very few motoneurons are labeled after injecting 20~l ofCTB-HRP in multiple sites along the muscle; they arefound scattered in a loose column in the ventral part of theMD subdivision.

The M. abductor auris longus originates on the sagittalcrest and ends on the lateral surface of the eminentiaconchae. It pulls the external ear caudally. The column oflabeled motoneurons is located ventrally and slightly medi­ally in the MD subdivision.

The M. rotator auris originates on the scutiform cartilageand ends on the caudo-medial surface of the eminentiaconchae. This muscle rotates the external ear medially andcaudally. Labeled cells are clustered in the ventral area ofthe MD subdivision.

Muscles that pull the pinna cranially. This group iscomposed of five muscles, whose motoneurons are found inthe INT and lateral areas of the MD subdivisions of the FN(Fig. 8).

The M. adductor auris superior is one of the largestmuscles associated with the pinna. The origin of thismuscle is on the dorsal surface of the scutiform cartilageand its insertion is in the cranio-medial margin of theauricular cartilage. Its action is to pull the auricle cranially.Injection of CTB-HRP into this muscle produced manylabeled cells in the lateral areas of the MD subdivision.

The M. frontoscutularis is a thick muscle that pulls theear cranially. Its origin is on the frontal bone, along thesupraorbital margin, and its insertion is on the scutiformcartilage, the cartilage of the ear, and the M. adductor aurissuperior. M. Frontoscutularis' motoneurons are locatedlaterally in the MD subdivision, overlapping with the moremedial motoneurons of the M. adductor auris superiorcolumn.

The M. adductor auris inferior originates on the orbitalligament and ends on the tip of the M. antitragicus. Thismuscle pulls the ear cranio-dorsally and. its motoneuronsare located ventro-laterally in the MD subdivision.

The M. corrugator superciLii Lateralis originates from theM. frontoscutularis and from the tendon lying just cranialof the external opening of the ear, and is inserted at the

Fig. 4. Cholera toxin B-horse radish peroxidase (CTB-HRP) labeled facial motoneurons after injectionsinto the M. levator auris longus. Both the somas and dendritic processes are darkly stained. An extendedsurvival time (96 hours) was used in this experiment.

605

longus) and ends on the concha. Its action is to flex theauricular cartilage. This muscle is innervated by tightlypacked motoneurons located dorsally in the MD subdivi­sion.

The M. transversus auriculi originates on the medialsurface of the concha and inserts on the auricular cartilage.It flexes the scapha medially, enlarging the auditory open­ing. Injection of CTB-HRP into this muscle produceslabeled motoneurons in the dorsal aspects of the MDsubdivision.

The M. tragicus medialis (cranial portion) flexes theconcha. Its origin is on the ventral end of the tragus, and itsinsertion is in the cranial surface of the concha. Labeledmotoneurons are found scattered in the ventral half of theMD subdivision. It was not possible to inject the caudalsubdivision of this muscle.

TheM. antitragicus originates at the caudal border of theantitragus and inserts on the tragus. This muscle constrictsthe external auditory opening. In the coronal plane, labeled

30fJm

A

caudo-Iateral angle of the eye. This muscle acts upon thepinna indirectly, pulling it cranially. The motoneuronsinnervating this muscle are located in the INT subdivision.

The M. frontoauricularis originates along the uppereyelid and it is inserted in the auricular cartilage, alongwith the M. abductor auris superior at the cranio-medialangle. This muscle consists of scattered groups of fibers (afeature that makes its study particularly difficult) that pullsthe ear cranially; it is also a weak adductor. Injection ofCTB-HRP in several locations into this muscle labeledmotoneurons in the INT subdivision only.

Muscles that change the shape of the pinnaThis group is composed of six muscles, innervated by

motoneurons that extend from the dorsal to the ventraledges of the MD subdivision and centered along its medio­lateral axis (Fig. 9).

The M. auricularis externus begins on the eminentiaconchae (distal to the insertion of the M. adductor auris

I NERVATIO OF PI A MUSCLES OF THE CAT

L.e. POPULIN AND T.e.T. YIN

caudal fibers of the M. adductor auris superior. It draws thecranial margin of the auricle proximally. The cells thatinnervate this muscle are found in the ventral portion ofthe MD subdivision.

Columnar organizationThe topographical organization of pinna motoneuron

pools described in the previous section is generally wellconserved along the rostro-caudal extent of the nucleus.Thus, when successive coronal sections are aligned, acolumnar pattern of organization for individual motoneu­ron pools is usually evident. To illustrate the columnar

(each 70 ~m thick), after injection ofCTB-HRP into individual musclesof the indicated groups. The broken lines indicate boundaries that couldnot be clearly identified. For details on subdivisions see the legend ofFigure 2 and the text. The scale bars, 1 mm, and the orientation is thesame as in Figures 2 and 3.

o 0 - 0o

Adductor Auris Medius

Intermedius Scutulorum

Auricularis Superior

Levator Auris Longus

cells are arranged in a ribbon-like fashion that extendsdorso-ventrally, slightly lateral in the MD subdivision.

The M. conchaeus externus constricts the concha. Boththe origin and insertion points of this muscle are located inthe concha. The origin is attached a short distance distal ofthe M. antitragicus, and the insertion is attached at a pointcaudo-dorsal of the origin. Motoneurons innervating thismuscle are found in the ventral part of the MD subdivision,although at more rostral levels some cells are displaced tothe center of the subdivision.

The M. helicis originates on the medial surface of theconcha and ends on the auricular cartilage, along with the

Fig. 5. Innervation to muscles that pull the pinna dorsally, dorsa­cranially, and dorsa-caudally. This and the following four figures arecamera lucida drawings of coronal sections of the facial nucleus atapproximately 25, 50, and 75% of its length (left to right) showing thelocation of single labeled motoneurons (e) from two adjacent sections

606

607

volume varied from 20-50 ~l, the number of labeledmotoneurons varied from 7-322. In part this correlation isto be expected since larger muscles, with larger volumes,may be innervated by more motoneurons (Buchthal, 1960).Injections of five muscles (M. frontoauricularis, tragicusmedialis, transversus auriculi, zygomaticus major, andsubmentalis) resulted in less than 20 labeled motoneurons;while six muscles (M. adductor auris superior, adductorauris medius, intermedius scutulorum, frontoscutularis,antitragicus, and rotator auris) resulted in more than 100labeled motoneurons.

In contrast to the large variability resulting from injec­tions into different muscles, there were very consistentresults from replications of injections into the same muscle.The consistency was evaluated on a subset of nine musclesthat were readily accessible for injection and resulted inmany labeled motoneurons (M. abductor auris longus,

Submentalis

Zygomaticus Major

Tragicus Lateralis

Depressor Conchae

Fig. 6. Innervation to muscles that pull the pinna ventrally and ventra-cranially.

organization along the rostro-caudal axis, we replicatedinjections of two muscles, the M. adductor auris medius andM. adductor auris inferior, whose motoneurons are seen inthe coronal plane in Figure 3, but we cut the sections in theparasagittal plane (Fig. 10). Labeled cells are found insimilar position along the dorso-ventral axis of the nucleusin bothsets of figures. Columns formed by smaller numbersof labeled cells such as those innervating the M. abductorauris brevis and M. frontoauricularis (not shown) are lesswell defined.

Variability in labeled motoneuronsIn spite of our efforts to use standardized procedures,

there was considerable variability in the intensity andnumber of motoneurons labeled following injections intodifferent muscles. This variability was only weakly corre­lated with the amount of CTB-HRP injected: while the

INNERVATIO OF PIN A MUSCLES OF THE CAT

Fig. 7. Innervation to muscles that pull the pinna caudally.

L.C. POPULIN AND T.C.T. YIN

Topographical organizationIt has been established in a variety of species that pinna

motoneuron pools are located in the medial areas of the FN(Papez, 1927; Courville, 1966; Martin and Lodge, 1977;Provis, 1977; Henkel and Edwards, 1978; Kume et al.,1978; Watson et al., 1982; Friauf and Herbert, 1985; Vidalet al., 1988). The topographical organization of these poolshas been examined in detail in both rat and bat (Friauf and

spread of HRP from the injection site into neighboringmuscles while underestimates could result from inadequatediffusion ofCTB-HRP within the muscle. Clearly, reducingthe spread of the labeling enzyme and maximizing thenumber of labeled motoneurons innervating each muscleare contradictory procedures. We feel it is more importantthat the labeled motoneurons resulting from a particularinjection originate only from the injected muscle, and notneighboring muscles, than that all of the motoneuronsinnervating that muscle be labeled. Thus, in minimizingthe possibility of overestimating, it is unlikely that allmotoneurons of a given muscle were labeled.

There was considerable variability in the numbers oflabeled cells following injections into different muscles, butrepeated injections into the same muscle invariably led tosimilar numbers of labeled cells, which were located in thesame part of the facial nucleus. If many, or few, labeledmotoneurons were seen after injection of a particularmuscle, then many, or few, respectively, were seen follow­ing a repeated injection.

Abductor Auris Longus

Rotator Auris

Abductor Auris Brevis

DISCUSSIONMethodological considerations

The chief limitation of this study is the possibility that wehave mis-estimated the number of motoneurons innervat­ing individual muscles. Overestimates could result from

adductor auris inferior, adductor auris medius, adductorauris superior, auricularis superior, depressor conchae,intermedius scutulorum, levator auris longus, and transver­sus auriculi) as well as five muscles (M. frontoauricularis,submentalis, tragicus medialis, transversus auriculi, andzygomaticus major) that had very few labeled motoneuronsfollowing the initial injection. Without exception, the loca­tion of the labeled motoneuron pools and the number oflabeled cells observed in the replications closely resembledthose in the original experiments. For example the replica­tions shown in Figures 3 and 10 resulted in 216 and 207labeled motoneurons for M. adductor auris medius and 73and 65 motoneurons for M. adductor auris inferior in theoriginal and replication experiments, respectively. We wereparticularly concerned with the muscles that resulted invery low numbers of labeled motoneurons so that severalreplications were made of those injections. For example, weinjected the M. frontoauricularis in four repeated experi­ments, in each case less than 20 cells were labeled. Simi­larly, in the other four muscles with few labeled motoneu­rons following the initial injection, we also obtained fewerthan 20 labeled cells in the replication, all consistentlylocated in the expected areas of the nucleus.

608

609

1990), possum (Provis, 1977), mouse (Komiyama et al.,1984), and rat (Watson et al., 1982; Semba and Egger,1986), the rostro-caudal axis of the facial musculature isrepresented along the latero-medial axis of the FN, and thedorso-ventral axis is represented along the dorso-ventralaxis of the nucleus.

Figure 11 is a composite picture of the cross section of theFN of the cat at approximately 50% of its length. Areas

Fig. 8. Innervation to muscles that pull the pinna cranially and cranio-dorsally.

Adductor Auris Inferior

Corrugator Supercilii Lateralis

Adductor Auris Superior

Frontoscutularis

Frontoauricularis

Herbert, 1985), but in the cat comparable studies arelacking.

The present results in the cat confirm the reports citedabove concerning the location of pinna motoneuron poolswithin the FN, showing that this subgroup of facial moto­neuron pools is arranged in a topographical fashion, whichis consistent with the somatotopic organization of theentire nucleus in other species. In monkey (Welt and Abbs,

INNERVATION OF PINNA MUSCLES OF THE CAT

•

"

Fig. 9. Innervation to muscles that change the shape of the pinna.

Antitragicus

Transversus Auriculi

Auricularis Externus

Tragicus Medialis

Conchaeus Externus

Helicis

1mm

R~

dorsal two thirds, and the latter the ventral one half of theMD subdivision. An exception to this organization seems tobe the innervation of the M. adductor auris inferior whichpulls the pinna cranio-dorsally and is located in the ventralhalf of the MD subdivision. Based on the dorsal componentin the line of pulling of this muscle, its motoneurons wouldbe expected to be located more dorsally, but the orientationof its muscle fibers (see Fig. 1, lower left) indicate that such

611

adductor auris inferior. Data are from injections made on the left andright side of the same animal; the photomicrograph in B is shownreversed.

Fig. 10. Cresyl violet stained parasagittal sections of the brainstemillustrating the rostro-caudal columnar organization of the motoneu­ron pools that innervate the (A) M. adductor auris medius and (B) M.

INNERVATION OF PINNA MUSCLES OF THE CAT

containing motoneurons that innervate muscles pulling intwo directions (e.g., dorso-caudal) are plotted in two differ­ent figures. The organization of these panels is based on theaction of the muscle groups which, in most cases, isconsistent with their somatotopic arrangement. FigurelIA,B shows the location of motoneuron pools that inner­vate muscles with dorsal and ventral components in theirdirection of pulling; the former occupy approximately the

•

•

•

ventre·cranially

ventrally

:::::: ventre-cranially

cranially

~ dorso-cranially

_. crania-dorsally

L.C. POPULI AND T.C.T. YIN

~ constricts

B

D

(el caudally and dorsa-caudally, (Dl cranially, dorsa-cranially, crania­dorsally, and ventro-cranially, and ofmotoneurons innervating musclesthat change the shape ofpinna (El.

muscle that pulls the external ear ventrally) are locatedexclusively in the VM subdivision.

Figure llC,D depicts the location ofmotoneurons inner­vating muscles that pull the external ear caudally and

dorsa-caudally

dorso-cranially

• caudally

II dorsa-caudally

,-0

E

A

c

a component may be minimal. Thus it is consistent with allother cranially pulling muscles (Fig. llD), whose motoneu­rons are located in the lateral portion of the MD subdivi­sion. The mOtoneurons of the M. depressor conchae (a

Fig. 11. Schematic representation of experimental findings. Gen­erallocation of motoneurons innervating muscles that pull the externalear (Al dorsally and dorsa-caudally, (Bl ventrally and ventro-cranially,

612

•

•

ERVATIO OF PI A MUSCLES OF THE CAT

cranially. Caudally pulling motoneurons are located medio­ventrally in the MD subdivision, and cranially pullingmotoneurons are located laterally in the MD and INTsubdivisions. Along the dorso-ventral axis of this lateralhalf of the MD subdivision, motoneurons that pull dorso­cranially (e.g., M. adductor auris medius) are located dorsalto those that pull cranio-dorsally <M. adductor auris infe­rior). Another apparent inconsistent finding is that moto­neurons that pull ventro-cranially (e.g., those innervatingthe M. zygomaticus major) are located more dorsally thanthose that innervate the M. adductor auris inferior, amuscle with a cranio-dorsal line of pull. However, M.zygomaticus major is located more cranially than perhapsany other muscle associated with the pinna (Fig. 1). Thusconsistent with the general somatotopic scheme, its moto­neurons are located laterally in the areas of the FN devotedto the pinna. Furthermore, a few M. zygomaticus majormotoneurons are located in the medial edge of the LATsubdivision. Motoneurons of two cranially pulling muscles,the M. frontoauricularis and M. corrugator supercilii latera­lis, are found in the INT subdivision; an observation thatalso fits within the general organizational scheme. Caudallypulling motoneurons are located medio-ventrally in the MDsubdivision, with dorso-caudally pulling motoneurons lo­cated more dorsally (Fig. llC).

Figure HE illustrates the location of motoneuron poolsinnervating the six muscles that change the shape of thepinna, which are arranged in a dorso-ventrally orientedribbon in the central region of the MD subdivision. Roughly,the dorsal one half of this area is occupied by motoneuronsof muscles that flex the external auditory structures, a,ndthe lower one half is occupied by motoneurons of themuscles that constrict the concha and the external auditoryopening. The topographical organization of these motoneu­ron pools is also consistent with the somatotopic arrange­ment of the musculature they innervate. That is, muscleswith a flexing action are located in the most distal parts ofthe pinna, and the motoneurons that innervate them arelocated in the dorsal one half of the MD subdivision, whilemuscles that constrict the external auditory structures arelocated more ventrally, and so are their respective motoneu­rons.

Friauf and Herbert (1985) concluded that such organiza­tion was not found among pinna motoneuron pools in theFN of the rat or the bat, which is surprising for the bat sinceit moves its pinnae very extensively. We found, however,that the location of four of the five motoneuron poolsstudied by Friauf and Herbert (1985) in the bat is verysimilar to the location of motoneuron pools innervatinghomologous pinna muscles in the cat. The same comparisonholds for only one motoneuron pool in the rat.

Rostrocaudally the FN seems to follow the columnarorganization observed in a variety of other motor systemssuch as the larynx (Yoshida et al., 1983), the tongue(Sokolov and Deacon, 1992), the trigeminal motor nucleus(Jacquin et al., 1983), and the spinal cord (Burke et al.,1977; Brink et al., 1979; Larnicol et al., 1982; Swett et al.,1986; Miller, 1987).

Overall, motoneuron pools within individual groups over­lap to different degrees. Those that innervate smallermuscles are not as tightly clustered as those that innervatelarger muscles. However there is little overlap among thefive different muscle groups. To some extent these observa­tions could have been favored by the design of our experi­ment, which minimizes the possibility of overestimatingthe number oflabeled motoneurons. As discussed above, it

613

is unlikely that all motoneurons innervating any givenmuscle are backfilled by our procedures. On the other hand,the consistency of results when the same muscle wasinjected repeatedly makes us believe that the overall pat­tern observed is genuine though perhaps not to the quanti­tative extent seen in our experiment. We conclude, there­fore, that the edges of the areas in Figure llA-E, althougharbitrary are likely to reflect the segregation of these poolswithin the confines of the nucleus. This is consistent withthe clear-cut topographic organization of pinna motoneu­ron pools in the FN of the bat (Friauf and Herbert, 1985).

ACKNOWLEDGMENTSThe authors are indebted to the staffof the Neurophysiol­

ogy Department Jo Anne Eckleberry, Joan Meister, andInge Siggelkow of the Histology Laboratory' Carol Dizack;Bob Klipstein, Ravi Kochhar, and Terry Stewart. We arealso thankful to Dr. John K. Harting and Dr. Philip H.Smith for helpful comments on an earlier version of thismanuscript. This work was supported by NIH grantDCOOIl6.

LITERATURE CITEDBrink, E.E., J.I. Morrell, and D.W. Pfaff (1979) Localization of lumbar

epaxial motoneurons in the rat. Brain Res. 170:23-41.Buchthal, F. (1990) The general concept of the motor unit. Res. Pub!. Assoc.

Res. Nerv. Ment. Dis. 38:3-30.Burke, RE., P.L. Strick, K. Kanda, C.C. Kim, and B. Walmsley (1977)

Anatomy of medial gastrocnemius and soleus motor nuclei in cat spinalcord. J. Neurophysio!. 40:667-680.

Calford, M.B., and J.D. Pettigrew (1984) Frequency dependence of direc­tional amplification at the cat's pinna. Hear. Res. 14:13-19.

Courville, J. (1966) The nucleus of the facial nerve: The relation betweencellular groups and peripheral branches of the nerve. Brain Re .1:338--J54.

Crouch, J.E. (1969) Text-Atlas of Cat Anatomy. Philadelphia: Lea andFebiger.

Friauf, E., and H. Herbert (1985) Topographic organization of facialmotoneurons to individual pinna muscles in rat (Rattus rattus) and bat(Rosettus aegyptiacus). J. Compo Neuro!. 240:161-170.

Henkel C.K., and S.B. Edwards (l978) The superior colliculus control ofpinna movements in the cat: possible anatomical connections. J. Compo

euro!. 1 2:763-776.Jacquin, M.F., R.W. Rhoades, H.L. Enfiejian, and M.D. Egger (1983)

Organization and morphology of masticatory neurons in the rat: Aretrograde HRP study. J. Comp. euro!. 218:239-256.

Komiyama, M., H. Shibata, and T. Suzuki (1984) Somatotopic representa­tion of facial muscles within the facial nucl~us of the mouse. BrainBehav. Evol. 4:144-151.

Kume, M., M. Uemura, K. Matsuda, R Matsushima, and N. Mizuno (1978)Topographical representation of peripheral branches of the facial nervewithin the facial nucleus: A.HRP study in the cat. eurosci. Lett. 8:5-8.

Larnicol, ., D. Rose, D. Marlot, and B. Duron (1982) Anatomical organiza­tion of cal intercostal motor nuclei as demonstrated by HRP retrogradelabeling. J. Physiol. (Paris) 78:198--206.

Martin, M.R, and D. Lodge (1977) Morphology of the facial nucleus of therat. Brain Res. 123:1-12.

Mesulam, M.·M. (1978) Tetramethyl benzidine for horseradish peroxidaseneurohistochemistry: A non-carcinogenic blue reaction product withsuperior sensitivity for visualizing neural afferents and efferents. J.Histochem. Cytochem. 26:106-117.

Miller, A.D. (1987) Localization of motoneurons innervating individualabdominal muscles of the cat. J. Compo eurol. 256:600-606.

Mu icant, A.D., J.C.K. Chan, and J.E. Hind (1990) Direction-dependentspectral properties of cat external ear: ew data and cross-speciescomparisons. J. Acoust. Soc. Am. 87:757-781.

Papez, J.W. (1927) Subdivisions of the facial nucleus. J. Comp. eurol.43:159-191.

Phillips, D.P., M.B. Calford, J.D. Pettigrew, L.M. Aitkin, and M.N. Semple

614

(1982) Directionality of sound pressure transformation at the cat'spinna. Hear. Res. 8: 13-28.

Populin, L.C., and T.C.T. Yin (1992) Topographical organization of themotoneuron pools innervating the muscles of the pinna in the facialnucleus of the cat. Soc. eurosci. Abstr.18:1194.

Provis. J. (1977) The organization of the facial nucleus of the brush·tailedpo sum (Trichosurus uulpecula). J. Comp. eurol. 172:177-188.

Rice, J.J .. B.J. May. GA Spirou, and E.D. Young (1992) Pinna-basedspectral cues for sound localization in cat. Hear. Res. 58:132-152.

Semba, K., and M.D. Egger (1986) The facial ''Motor'' nerve of the rat:control of vibrissa! movement and examination of motor and sensorycomponents. J. Compo eurol. 247:144-158.

Siegmund, H., and G. Santibaiiez-H (1981) Effects of motor degeneration ofthe external ear muscles on the audio-visual targeting reBex in cats. Acta

eurobiol. Exp. 41:1-13.Siegmund H., and G. Santibaiiez-H (1982) Effector pattern of the audio­

visual targetingreBex in cats. Acta eurobiol. Exp. 42:311-326.Sokoloff, A.J., and T.W. Deacon (1992) Musculotopic organization of the

hypoglossal nucleus in the cynomolgus monkey, Macaca (ascicularis. J.Comp. eurol. 324:81-93.

Swett, J.E., R.P. Wilkholm, RH.l. Blanks, A.L. Swett, and L.C. Conley(1986) Motoneurons of the rat sciatic nerve. Exp. eurol. 93:227-252.

L.e. POPULIN AND T.e.T. YIN

Trojanowski, J.Q., J.O. Gonatas, and N.K. Gonatas (1982) Horseradishperoxidase (HRP) conjugates of cholera toxin and lectins are moresensitive retrogradely transported markers than free HRP. Brain Res.231:33-50.

Vidal, P.-P.,P.J. May, and R. Baker (1988) Synaptic organization of thetectal-facial pathways in the cat. L Synaptic potentials following collicu­tar stimulation. J. Neurophysiol. 60:769-797.

Watson, C.R.R., S. Sakai, and W. Armstrong (1982) Organiz.a.tion of thefacial nucleus in the rat. Brain Behav. Evol. 20: 19-28.

Welt, C., and J.H. Abbs (1990) Musculotopic organization of the facial motornucleus in Macaca (ascicularis: A morphometric and retrograde tracingstudy witb cholera toxin B-HRP. J. Comp. eurol. 29I:621-{;36.

Wiener, F.M., R.R. Pfeiffer, and S.. Backus (1966) On the sound pressuretransformation by the bead and auditory meatus of the cat. ActaOtoJaryngoL 61:255-269.

Yoshida, Y., M. Hirano, T. Shin, and T. Kanaseki (983) Topographicarrangement of motoneurons innervating the suprahyoid and infrahyoidmuscles: A horseradish peroxidase study in cats. Ann. Otol. Rhinol.Laryngol. 92:259-266.

Zook, J.M., HA Patterson, and J.C. Middlebrooks (981) Topographicorganization of motor neurons innervating pinna muscles in the cat. Soc.

eurosci. Abstr. 7:54.

!