The facial “motor” nerve of the rat: Control of vibrissal movement and examination of motor and sensory components

THE JOURNAL OF COMPAl3ATIVE NEUROLOGY 247~144-158 (1986)

The Facial “Motor” Nerve of the Rat: Control of Vibrissal Movement and Examination of Motor and Sensory

Components

KAZUE SEMBA AND M. DAVID EGGER Department of Anatomy, UMDNJ-Rutgers Medical School, Piscataway,

New Jersey 08854

ABSTRACT Rhythmical whisking of the mystacial vibrissae at about 7 Hz during

exploration is one of the most conspicuous behavioral patterns in the rat. To identify the final common pathway for vibrissal movement, individual motor branches of the facial nerve, including the posterior auricular, temporal, zygomatic, buccal, marginal mandibular, cervical, stylohyoid, and posterior digastric branches, were cut, either singly or in various combinations. We found that vibrissal movement could be abolished only by transection involving the buccal branch and the upper division of the marginal mandibular branch.

To trace back the central origins of the buccal and marginal mandibular, as well as the other branches of the facial nerve, all distal to the stylomastoid foramen, horseradish peroxidase (HRP) was applied to the cut proximal ends of these individual branches. The retrograde HRP labelling in the facial motor nucleus revealed topographical representation of these branches in which the buccal and marginal mandibular branches were represented laterally. The stylohyoid and posterior digastric branches originated from cells in the suprafacial nucleus. Consistent with earlier observations with intramuscular HRP injections, the motoneuronal population devoted to vibrissal movement did not seem to be substantially larger than that for other facial movements.

An additional examination was made of the labelled afferent component of the facial motor nerve. We confirmed and extended previous findings that none of the above facial motor nerve branches, except the posterior auricular branch, contained a significant number of afferent fibers originating from the geniculate ganglion, the sensory ganglion of the seventh nerve. In addition, no labelling was seen in the mesencephalic trigeminal nucleus or trigeminal ganglion. These findings, in combination, suggest that, with the exception of the posterior auricular branch, all the facial motor nerve branches, including those involved in vibrissal movement, are almost en- tirely efferent.

Key words: HRP transport, facial nerve branches, facial nucleus, geniculate ganglion

During exploration, rats move the mystacial vibrissae back and forth at a rate of about 7 Hz. This rhythmical movement of the vibrissae is synchronized with sniffing as well as rhythmical movements of the nose and head (Welker, ’64) and represents one of the most conspicuous behavioral patterns in this species. In addition to this “whisking” movement, many rats display tremorlike movement of the vibrissae, which occurs at about 9 Hz, and in a smaller amplitude during behavioral arrest (Semba et al., ’80;

Semba and Komisaruk, ’84). Although the vibrissae have been recognized as constitut-

ingan important sensory organ to guide rats’ behavior (e.g.,

Accepted November 8, 1985. Address reprint requests to K. Semba, Division of Neurological

Sciences, University of British Columbia, Vancouver, B.C. V6T 1W5 Canada.

0 1986 ALAN R. LISS, INC.

RAT FACIAL MOTOR NERVE 145

Vincent, '12; Schiffman et al., '70), and much research has been conducted on the sensory input from the vibrissae at different levels along its ascending pathways (e.g., Zucker and Welker, '69; Welker, '71; Waite, '73; Woolsey et al., '75; Van der Loos, '76; Belford and Killackey, '79; Arvidsson, '82), relatively little is known about the motor control of vibrissae. For example, vibrissal movement is actuated by the muscles around the muzzle, which have been thought to be innervated by the facial nerve (FN). Of several FN branches, the zygomatic branch has been shown to control vibrissal movement in the North American opossum (Dom et al., '73). The zygomatic-orbital and superior buccolabial branches control vibrissal movement in the brush-tailed possum (Provis, '77). However, the FN branch or branches controlling vibrissal movement are not exactly identified in the rat, the species in which most of the research on sensory input from the vibrissae has been conducted.

In the present study, we investigated this question by examining the effects of cutting individual FN branches, as well as their subdivisions, on vibrissal movement in behav- ing rats. The results of this experiment indicate that vibrissal movement is controlled by the buccal branch and the upper division of the marginal mandibular branch in the rat.

The identification of the FN branches controlling vibrissal movement led us to our next question concerning the central origin of these branches. This question was investigated in the more general framework of the representation of all individual FN branches in the facial motor nucleus. This study was also a prerequisite to a study on the morphology and physiology of motoneurons involved in vibrissal movement.

In the facial nucleus, several subnuclei have been recognized in Nissl-stained material, and these subnuclei have been correlated with peripheral FN branches in a variety of mammalian species on the basis of various retrograde neuronal changes following nerve cuts (Papez, '27; Cour- ville, '66b; Dom et al., '73; see also Vraa-Jensen, '42, for review of early work), motor fiber and end-plate degeneration following lesions of the facial nucleus (Szentagothai, '481, and antidromic field potentials (Kitai et al., '72; Mar- tin and Lodge, '77). With the advent of the horseradish peroxidase (HRP) technique, more precise representations of peripheral branches have been revealed in the cat (Kume et al., '78) as well as in the brush-tailed (Provis, '77) and North American opossum (Dom, '82).

More recently, musculotopic organization has been revealed in the facial motor nucleus following HRP injections into various facial muscles in the rat (Watson et al., '82; Hinrichsen and Watson, '84) and mouse (Ashwell, '82; Ko- miyama et al., '841, as well as in the monkey (McGuiness, '81; Welt et al., '84). However, these findings could not be directly used to interpret our results on the effects of FN branch cuts on vibrissal movement, because, in the rat, the exact relationship between the peripheral branches and the facial muscles innervated by them is not known. We therefore re-examined the central origins of peripheral FN branches by applying HRP directly to cut ends of these individual branches, followed by the examination of retrograde HRP labelling in the facial nucleus.

Although the FN is primarily an efferent nerve, it is also known to contain some afferent fibers. For example, in previous studies (Semba et al., '83, '84) using the anterograde HRP transport technique in the rat, we found that about 30% of the cells in the geniculate ganglion (GG), the

sensory ganglion of the seventh nerve, contributed fibers to the posterior auricular branch of the FN. In the cat, these fibers have been shown to convey sensory input from the inner surface of the ear (Boudreau et al., '71). In contrast to the posterior auricular branch, however, the other, more distal branches, including those controlling vibrissal movement, contained few afferent fibers originating from GG cells (Semba et al., '84). In our earlier study, the stylohyoid and posterior digastric branches of the FN were not included. These branches leave the FN motor trunk proximal to the posterior auricular branch, at the stylomastoid foramen. In the present study, therefore, we report the results with the stylohyoid and posterior digastric branches, in conjunction with those with the other branches of the FN. In addition, we also looked for transganglionic/anterograde labelling in the brainstem, cervical spinal cord, and trigeminal ganglion, following HRP applications to different FN branches.

To summarize, the aim of the present study was to identify the peripheral branch or branches of the FN that are involved in the control of vibrissal movement and to examine the efferent and afferent components of these branches as well as the other FN branches, all distal to the stylomastoid foramen in the rat. Preliminary observations have been published (Semba, '84; Semba and Egger, '84).

MATERIALS AND METHODS Animals

Adult Sprague-Dawley rats, mostly male, were used. Fif- teen rats were dissected either under deep anesthesia or after they died of overdose in order to study the peripheral branches of the FN. A second group of 28 rats, weighing from 250 to 350 g, was used for behavioral observations on the effects of cutting peripheral FN branches on vibrissal movement, and a third group of 80 rats, typically weighing about 200 g (range: 140-300 g), was used both for additional behavioral testing and for HRP transport labelling. A fourth group of three rats served as control for HRP application and labelling.

Transection of FN branches The procedure for cutting individual FN branches is de-

scribed as part of the HRP application procedure described below. In the rats to be tested behaviorally without HRP application, the skin was sutured together immediately after transection. In these rats, if the initial nerve cut did not affect vibrissal movement, subsequent surgery was performed to cut additional branch(es).

Behavioral observations For the 29 rats in the behavioral observation group, ob-

servations on vibrissal movement were conducted repeat- edly between 1 day and 10 months following nerve transection. In the group of 83 rats used for HRP transport, behavioral observations were conducted between 1 and 5 days following nerve transections. To observe vibrissal movement, individual rats were placed on a painted black box that was 12 x 12 x 30 cm in size. This box was placed on a large sheet of black paper, which provided a good background for the white vibrissae. Putting a rat on the box usually elicited a prolonged (several minutes or longer) period of rhythmical whisking of the vibrissae, along with an exploratory sniffing. In addition, various novel stimuli, such as the smell of food or the sound of clinking keys, were used to induce vigorous vibrissal movement.

146 K. SEMRA AM) M.D. EGGER

phosphate buffer (final pH adjusted to 7.3) at 4°C with agitation. After 3-5 days, following one to two changes of solution, the decalcified tissue was transferred to 30% sucrose in the same buffer at 4°C for 1-2 days. The tissue was cut mostly horizontally, but some sagittally, on a cryostat into 10-pm sections that were immediately mounted on 2% gelatinized slides.

Both brain and GG sections were histochemically treated by using tetramethylbenzidine as a chromogen according to the method of Mesulam ('78). Following the histochemical reaction, brain sections were mounted on 2% gelatinized slides and air-dried overnight. Geniculate ganglion sections, which had been reacted on slide, were simply air- dried. Alternate brain sections and all GG sections were counterstained with thionin according to Adams ('80). All the sections were cleared in xylene and coverslipped with an embedding medium.

Histological analysis The sections were examined and photographed in a Zeiss

or Nikon microscope. Labelled and unlabelled cells in the facial nucleus were counted in all the alternate, thionin- stained sections that contained the facial nucleus. These cell counts were then corrected for split cell errors according to the method of Konigsmark ('70).

RESULTS The peripheral branches of the facial motor nerve The seventh cranial nerve is composed of two nerves: the

intermediate nerve and the FN. The intermediate nerve is often referred to as the sensory root, and contains both sensory (gustatory and tactile) and parasympathetic fibers. The sensory fibers have their cell bodies in the GG. The FN, representing the motor root, arises from the facial nucleus in the rostra1 medulla. After exiting the brainstem from its ventrolateral aspect, the FN transverses the temporal bone in which it gives off motor fibers to innervate the stapedius muscles (e.g., Rhinehart, '18). These fibers to the stapedius are not included in the present study.

Emerging from the cranium via the stylomastoid foramen, the FN divides into several branches, and the present study concerned these branches. The branches included (1) the posterior auricular, temporal, zygomatic, buccal, marginal mandibular, and cervical branches, innervating various superficial muscles of the face and head, and (2) the stylohyoid and posterior digastric branches, innervating small deeper muscles, i.e., the stylohyoid muscle and the posterior belly of the digastric muscle, respectively (Fig. 1; nomenclature according to Greene, '35). They were identified during the initial stage (dissection) of the study by electrical stimulation to induce muscle contractions, and later by inspection in a dissection microscope.

Each of the peripheral branches consisted of a single nerve, except for the cervical and temporal branches, which were composed of a few smaller nerves. In addition, the marginal mandibular branch had two easily recognizable divisions: the upper division, which innervated muscles in and near the mystacial vibrissal pad, and the lower division, which innervated the mentalis muscle. Although the pattern of secondary branching was somewhat variable among individual rats, that of primary branching, shown in Figure 1, seemed constant.

We were unable to identify the "lower zygomatic branch" illustrated by Greene ('35; her Figs. 150 and 151). Instead, we observed that two small nerves of the trigeminal origin

Surgery and HRP application HRP was applied to the posterior auricular (n = 17),

temporal (n = 41, zygomatic (n = 71, buccal (n = 9), marginal mandibular (n = 71, cervical (n = 81, and combined stylohyoid and posterior digastric branches (n = 4). The last two branches were combined because they originated from a common stem and their limited lengths made it technically difficult to treat them separately. In addition, HRP was appIied to all the branches (n = 81, temporal, zygomatic, buccal, marginal mandibular, and cervical branches (n = 71, buccal and marginal mandibular branches (n = 31, or the upper (n = 3) or the lower division (n = 3) of the marginal mandibular branch.

Rats were pretreated with atropine sulfate (10 mg/ml, 0.3-0.4 muanimal, i.pJ and Taractan (chlorprothixene, a tranquilizer, Roche, 0.1-0.15 ml, i.p.) and anesthetized with Ketaset (ketamine hydrochloride, Parke Davis, 0.1-0.15 ml, i.p., supplemented as needed). The peripheral FN branches were exposed by making an incision below an ear and retracting tissues including the lacrimal and parotid glands and the external jugular vein. For the stylohyoid and posterior digastric branches, located deeper, the sternomastoid muscle was also retracted. Exposed FN branches were cut as proximally as possible, and distilled water was applied for 2-5 minutes. The proximal cut end was then mounted on a small plastic sheet, and after cleaning excess water, a few flakes of HRP (Sigma, type VI) was applied to the stump, followed by a saturated HRP solution in 2% di- methyl sulfoxide soaked in a small piece of Gelfoam for 1- 2 hours. Occasionally a small amount of Vaseline was used as an insulator to prevent spread of HRP to adjacent tissue. After HRP application, all the excess HRP was cleaned with saline and the incision was sutured. All HRP applications were unilateral.

To examine the effects of possible leakage of HRP onto adjacent tissue including nerves, three rats were used. In two of the three, all the branches were exposed unilaterally and mounted onto a small plastic sheet without transection. Horseradish peroxidase was applied to the branches, as well as to adjacent tissue, for 2 hours, followed by washing with saline. In a third rat, in order to examine labelling resulting from possible spill of HRP from the plastic sheet to adjacent tissue HRP was placed for 2 hours on the plastic sheet positioned over all FN branches, which were exposed but uncut.

Histochemical reaction After 1-5 days (mostly 2 days for retrograde transport,

and 4 days for anterogradeltransganglionic transport), rats were overdosed with Nembutal and perfused intracardially with (1) 50-100 ml of saline containing 20 units/ml of hep- arin, and 0.04 mg/ml of Xylocaine at 37"C, (2) 500-1,000 ml of a fixative containing 2% glutaraldehyde and 1% para- formaldehyde in 0.1 M sodium phosphate buffer at room temperature, and (3) 500 ml of 10% sucrose in 0.1 M buffer at 4°C.

Following perfusion, the brain was removed from the skull. In addition, the temporal bone containing the GG was removed in the rats in which the GG was to be examined. The brains were placed in 10% sucrose in 0.1 M phosphate buffer (pH 7.3) at 4°C overnight, and cut into 50- pm coronal sections on a freezing microtome, which were collected in 0.1 M phosphate buffer.

The tympanic bone, containing the GG, was placed in 0.1 M ethylenediaminetetraacetic acid (EDTA) in 0.1 M sodium


* . . .

Fig. 1. Drawing of the peripheral branches of the facial nerve distal to the stylomastoid foramen in the rat. P AUR: posterior auricular; TEM: temporal; ZYG: zygornatic; BIJC: buccal; MAR MAN marginal mandibular; KJ): upper division; GI: lower division; CER: cervical; STY stylohyoid; P DIG: posterior digastric.

(confirmed by the presence of anterograde HRP labelling in the trigeminal ganglion as well as the absence of retrograde labelling in the facial nucleus) followed, in combination, a course similar to that illustrated by Greene for the “lower zygomatic branch.”

The effects of FN branch cut on vibrissal movement To identify the branches of the FN that contain efferent

fibers innervating the muscles actuating vibrissal movement, individual FN branches were cut either singly or in various combinations, and their effects on vibrissal movement were examined behaviorally in 108 rats. All the transections were unilateral. The results are summarized in Table 1.

Table 1 indicates that vibrissal movement could be blocked only when cut branches included both the buccal branch and the upper dvision of the marginal mandibular branch. The effects were unilateral, and the movement on the contralateral side appeared to be unaffected, serving as control. Cutting either the buccal branch or the upper marginal mandibular branch alone did not appear to affect vibrissal movement; whisking movement was bilaterally synchronous and apparently of the same amplitude in these rats, indistinguishable from that seen in unoperated rats. The transection involving all other branches, either singly or in combination, did not appear to affect vibrissal movement. It seemed therefore that cutting both the buccal branch and the upper division of the marginal mandibular branch was necessary and sufficient to abolish ipsilateral vibrissal movement completely.

The disappearance of vibrissal movement was apparently permanent. We observed no signs of recovery in the rats examined up to 10 months after nerve cut.

Efferent fibers The facial nucleus. The facial nucleus measured about

2.3 mm mediolaterally, 1.7 mm dorsoventrally, and 1.5 mm rostrocaudally in its maximum extent. Figure 2A shows a thionin-stained coronal section through the middle portion of the facial nucleus, in which five subnuclei previously described by Martin and Lodge (’77) may be recognized, including the lateral (L), dorsal (D), intermediate (I), ventromedial (VM), and medial (M). Dorsal to the facial nucleus lay a small group of cells, the suprafacial nucleus, also referred to as the accessory facial nucleus.

Consistent with the observations by Martin and Lodge (’77), the five subnuclei had somewhat different rostrocaudal extents: L and D were located most caudally, M most rostrally. All the subnuclei had boundaries that were usually discernible at most levels. The suprafacial nucleus was seen at intermediate to rostral levels of the facial nucleus, as well as in the reticular formation rostral to the facial nucleus.

TABLE 1. The Effects of Transection (XI of Peripheral Branches of the Facial Nerve on Vibrissal Movement

Facial nerve branches cut Margmal mandibular Stylohyoid and

Posterior posterior No. Vibrissal Buccal U L Cervical auricular Temporal Zygomatic digastric of rats movement

X X X X X X X X X X

x x X X X X x x X X X x x X X x x X X x x X X x x X

X X X X X

X X

x x X X X x x X

X X X X X X

X X X X X

X X

X

X 8 Absent 10 (3)’ Absent 1 Absent l (1) Absent 1 Absent I Absent 5 Absent l(1) Normal 2 Normal 2 Normal

10 1 8 5

X 4 (3) 6

X 3 8

17 4 7

Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal ~ .~~~~~

X 6 Normal ’Numbers in parentheses indicate the number of rats in which the nerve cuts were performed sequentially, in two or three stages. U: upper division; L: lower division.

148 K. SEMBA AND M.D. EGGER

Fig. 2. A. A coronal section through the intermediate level of the facial nucleus, stained with thionin. As indicated, the facial nucleus can be di- vided into five subnuclei including the lateral n), dorsal (D), intermediate (I), ventromedial (VM), and medial (M). The suprafacial (S7) nucleus is also seen dorsal to the main facial nucleus. B. A coronal section through the facial nucleus at the level comparable to that in A, and in the same orientation, following HRP application to all branches of the facial nerve distal to the stylomastoid foramen shown in Figure 1. HRP labelling is seen in all the subnuclei of the facial nucleus as well as in the suprafacial nucleus. The section was counterstained with thionin. Scale bars: 0.5 mm.

Estimates of the total neuronal populations in each of the iubnuclei are presented in Table 2. The average number of ieurons in the facial nucleus in 19 rats was 3,512 f 543 mean f S.D.) (corrected for split cell errors according to (onigsmark's "701 method). This is in close agreement vith the corrected total cell estimate (3,350 5 133, mean 5 LD., n = 6) reported by Martin et al. ('77). (Watson et al. '821 reported an estimate of 5,300-5,800 cells. Their cell :ounting method was unspecified, but their count agrees ,ather closely with our uncorrected count, 5,106-790, as vell as that of Martin et al. "771.) Interestingly, Martin et tl. ('77) also reported that the number of facial motoneu- .anal axons was 5,353 (n = l), which agrees with their and tlso our total cell population before correction for split cell :rrors. In the mouse, Ashwell ('82) reported a corrected tverage total cell estimate of 2,027, while Shimozawa ('75) ,eported an average of 3,215 motor fibers in the FN root. Labelling following HRP application to individual FN

iranches. Following HRP application to the FN trunk listal to the stylomastoid foramen (including all the iranches shown in Fig. l), labelled cells were seen in all he subnuclei of the facial nucleus as well as in the supra- acial nucleus (Fig. 2B). On the other hand, following HRP ipplications to individual branches, labelled cells were seen n restricted areas in a topographic manner as described ielow. The results are summarized in Table 3. Following HRP application to the posterior auricular

ranch alone, most labelled cells were seen in M, and some n VM (Fig. 3A). In the ventral half of M, almost all of the ells were labelled. Cells labelled by HRP application to the Iosterior auricular branch accounted for up to 15% of total ells in the facial nucleus. Labelling following HRP application to either the tem-

iora2 (Fig. 3B) or the zygomatic branch (Fig. 3C) was similar md localized dorsally in the facial nucleus, i.e., in D, as vell as in the dorsal regions of L, I, VM, and M. A minor lifference was that cells labelled by HRP application to the emporal branch were more or less evenly distributed in he above regions, whereas cells labelled by HRP applica- ion to the zygomatic branch tended to be concentrated aterally (see Table 3). Labelled cells accounted for up to 6% md 5% of total facial nuclear cells, respectively, following B P applications to the temporal and the zygomatic branch. Following HRP application to the buccal branch, up to 17% of the cells in the facial nucleus were labelled. A najority of these cells were found laterally in the facial iucleus, particularly in L, I, and D, although some labelled :ells were seen in the dorsal portion of M (Fig. 3D). Labelling following HRP applicatioon to the marginal

nandibular branch was similar to that seen following HRP tpplication to the buccal branch, except that labelled cells ended to be located slightly more ventrally (Fig. 3E). The abelled marginal mandibular cells accounted for up to 26% )f the cells in the facial nucleus. When HRP was applied separately to the upper and the

dower division of the marginal mandibular branch, different patterns of labelling were seen. Application of HRP to the upper division resulted in labelling (Fig. 4A) similar to that seen following HRP application to the buccal branch (Fig. 3D), whereas application of HRP to the lower division resulted in labelling of cells mostly in the ventral aspect of I (Fig. 4B), but lateral to the labelling seen following HRP application to the cervical branch (see below). The number of labelled cells was also different between the upper and


Fig. 3. Labelling in the facial nucleus following HRP application to individual branches of the facial nerve innervating superficial facial muscles. The sections were stained with thionin. All these coronal sections were through the rostrocaudal levels of the facial nucleus comparable to that

shown in Figure 2, except for the section shown in C, which was through a slightly more caudal level. Medial, right; dorsal, top. A posterior auricular; B: temporal; C: zygomatic; D buccal; E: marginal mandibular; F: cervical. Scale bar: 0.5 mm.


TABLE 2. The Total Number of Cells (Mean f S.D., n = 19) in Each of the Subnuclei of the Facial Nucleus

Total cell estimate Total cell estimate % Total cells in Subnucleus before correction' after correction2 the facial nucleus

Dorsal 450 i 94 310 f 65 8.8 Lateral 1,632 ? 222 1,122 k 153 32.0 Intermediate 1,590 f 308 1,094 k 212 31.1 Ventromedial 140 k 34 96 23 2.7 Medial 1,294 k 242 890 f 166 25.3 Combined 5,106 k 790 3,512 k 543 100.0

k

'Total cell estimate = 2 X c n , , where k is the total number of alternate sections containing a given

subnucleus, and n, the number of cells in that subnucleus in the i" section. 'Total cell estimate after correction for split cell errors according to the method of Konigsmark ('70).

i=O

TABLE 3. The Pattern of Labelling in the Facial Nucleus Following HRP Application to Individual Peripheral Branches of the Facial Nerve

Facial motor subnucleus' q%, Maximum total labelled cells Facial motor

nerve branch No. Inter- Ventro- in the facial exposed to HRP of rats Dorsal Lateral mediate medial Medial nucleus

Posterior auricular Temporal Zygomatic Buccal Marginal mandibular Cervical Stylohyoid and posterior digastric Combined

17 + ++++ 4 + + + + + + 7 +++ + + + + 9 +++ ++if + i t t + 7 + + + + + + + t + 8 + ++++ + 4

56 ++++ ++++ ++++ ++++ ++++

15 6 5

37 26 4 0

93

'+, Less than 10% of the total cells labelled in a given subnucleus; + +, 10-30% of total cells labelled + + +, 30-50% of total cells labelled; + + + +, more than 50%) of total cells labelled; no sign is indicated where no labelled cells were seen.

lower divisions; the number of cells labelled following HRP application to the lower division was approximately 30% of that following HRP application to the upper division.

Cells labelled following HRP application to the cervical branch were fewer in number than with any other single branch, accounting for only up to 4% of total facial nuclear cells. These cells were located medially to those labelled with the buccal or the mandibular branch, i.e., in VM and, less densely, in apposing border regions of I and M (Fig. 3F).

When HRP was applied to the stylohyoid and the posterior digmtric branches in combination, labelled cells were not seen in the facial nucleus but were seen in the suprafacial nucleus (Fig. 5). Rostrally, labelled cells could be followed dorsally into the reticular formation, becoming fewer in number, shifting dorsolaterally, and crossing the descending limb of the facial nerve root. Labelled cells were seen in sections as far rostrally as near the caudal tip of the motor trigeminal nucleus (Fig. 6).

Data that confirm the above results were provided by the rats in which HRP was applied to various combinations of individual branches (see Materials and Methods for specific combinations).

Although the axonal transport of HRP does not usually reveal the entire morphology of individual neurons, relatively good filling was obtained in some rats. For example, in one rat in which HRP was applied to both buccal and upper marginal mandibular branches, labelled facial motoneurons appeared large, with their longest dimensions ranging from 30 to 45 pm, and displayed multiple dendrites (Fig. 7). At the genu of the FN in the same rat, a small bundle of labelled axons approached the midline and then coursed back to the genu, forming a flat loop (not illustrated). Such loops were seen in some additional rats in

which HRP was applied to the posterior auricular, buccal, or combined stylohyoid and posterior digastric branches. Similar observations have been made by Martin and Mason ('77), but only with the stylohyoid and posterior digastric branches. Nishi ('651, on the other hand, observed (with a myelin staining method) that some fibers in the FN cross the midline, entering the genu of the contralateral side. Similar looping toward the midline has also been observed in the axons of trigeminal motoneurons (Jacquin et al., '83).

Virtually all labelling seen following HRP applications to FN branches was ipsilateral; only a few cells were labelled in the contralateral facial nucleus (two cells in one rat following HRP application to all the branches, and one cell in another rat following HRP application to the buccal branch).

In the two control rats in which HRP was applied to all exposed but uncut branches as well as adjacent tissues for 2 hours, some labelling was seen ipsilaterally in the cervical ventral horn, dorsal nucleus of the vagus, nucleus ambiguus, facial nucleus, suprafacial nucleus, and motor and mesencephalic trigeminal nuclei. Unlike the dark labelling following HRP application to cut branches, the labelling in these control rats was mostly faint. In a third control rat in which HRP was put on a small plastic sheet placed on all exposed but uncut branches for 2 hours, no labelled cells were seen in the brainstem or the spinal cord.

Afferent fibers HRP was applied to the posterior auricular branch (n =

3), the temporal, zygomatic, buccal, marginal mandibular, and cervical branches (n = 2), and stylohyoid and posterior digastric branches (n = 2). Anterogradehransganglionic labelling was examined in the GG, brainstem, upper cervical cord, and trigeminal ganglion.

RAT FACIAL MOTOR NERVE

Fig. 4. Labelling in the facial nucleus following HRP application to the upper (A) and the lower (B) division of the marginal mandibular branch. The section was counterstained with thionin. Medial, right; dorsal, top. Scale bar: 0.5 mm.

The geniculate ganglion. As shown in Figure 8 (see also Rhinehart, '18; Bruesch, '44), the GG consisted of two parts: the main GG, and the much smaller dorsal GG. Following HRP application to the posterior auricular branch, up to 23% of the cells were labelled ipsilaterally in both main and dorsal GG (Fig. 9). Following HRP application to the temporal, zygomatic, buccal, marginal mandibular, and cervical branches, less than 0.1% of cells were labelled in the main GG, and none in the dorsal GG. These results were comparable to those obtained in our previous study (Semba et al., '84), and are summarized in Table 4.

When HRP was applied to the stylohyoid and posterior digastric branches, less than 0.3% of GG cells, all in the main GG, were labelled (Table 4).

The brainstem and spinal cord. Following HRP application to the posterior auricular branch, some labelled fibers were seen to traverse the ipsilateral spinal tract of the trigeminal nerve and enter the dorsal half of the ipsilateral nucleus interpolaris of the spinal tract of the trigeminal

Fig. 5. Labelling seen in the suprafacial nucleus following HRP application to combined stylohyoid and posterior digastric branches. An arrow indicates a process of a labelled cell entering the facial nucleus. The section was counterstained with thionin. 7: the main facial nucleus. Scale bar: 0.5 mm.

nerve. No such labelling was seen following application of HRP to any other FN branch. No labelling was seen in the upper portion of the cervical spinal cord following HRP application to any FN branch.

In all the HRP applications we conducted, no labelling was seen in the mesencephalic trigeminal nucleus in its entire extent from the midbrain to the pons.

The trigeminal ganglion. The ipsilateral trigeminal ganglion was examined in the two rats in which HRP was applied to all the FN branches distal to the stylomastoid foramen. No labelling was seen.

DISCUSSION Peripheral branches of the facial "motor" nerve Examining the FN distal to the stylomastoid foramen in

the rat, we have identified five branches (the posterior auricular, temporal, zygomatic, buccal, marginal mandibular, and cervical) innervating various superficial muscles of the face and head, and two branches (the stylohyoid and posterior digastric) innervating deeper muscles. Although these observations are in general agreement with those of earlier studies in rat (Greene, '35; Hebel and Stromberg, '76; Martin and Lodge, '77) and mouse (Komiyama et al., '841, there are some discrepancies.

First, consistent with the observation by Hebel and Stromberg ('76) in the rat as well as that by Komiyama et al. ('84) in the mouse, we did not identify the "lower zygomatic branch" illustrated by Greene ('35). Some of our HRP experiments instead suggested that the nerve identified as such was a branch of the sensory trigeminal nerve. Such anastomosis between the facial and trigeminal nerves is rather common (e.g., Garin, '65), making the identification of smaller nerves potentially difficult unless supplemented by, for example, the HRP tracing method.

A second discrepancy concerns the cervical branch, which innervates the platysma. Hebel and Stromberg ('76) did not mention this branch. Greene ('35) described and illustrated it as a single nerve branching from the marginal mandibular branch. In agreement with Martin and Lodge ('77), we observed that the cervical branch consisted usually of two to three small nerves, rather than one, and that the site of their origins from the marginal mandibular branch were not as distal as illustrated by Greene ('39, but located more proximally, near the parotid glands. Consistently, Komi- yama et al. ('84) illustrated the cervical branch in the mouse as formed by three nerves near the parotid glands. How- ever, these authors also identified a fourth cervical nerve that supplies the rostral part of the platysma. Since we did not see this nerve in our rats, this difference may represent a species difference.

The pattern of ramifications of the facial motor nerve has been illustrated in several mammalian species. The pattern seen in rodents, described above, appears to be similar, in general, to that in cat (Papez, '27) and dog (Vraa-Jensen, '42). Somewhat different patterns have been described in the North American (Dom et al., '73) and the brush-tailed opossum (Provis, '77). In these species, the zygomatic or the zygomatic-orbital branch extends far more rostrally than in the other species, and this branch provides additional supply to the nasolabial muscles, which, in the other species, are innervated by the buccolabial branch alone. This rostral extension of the zygomatic branch seems to reflect the fact that the opossum has a rather elongated or pointed snout compared to rodents, dog, and cat.

Topographic representation of the peripheral branches of the facial nerve

The facial nucleus. We found that the peripheral FN branches innervating the superficial facial muscles (i.e., all the FN branches distal to the stylomastoid foramen, exclud- ing the stylohyoid and posterior digastric branches) are topographically represented in the facial motor nucleus, with some overlaps between particular branches. The to- pography was seen in such a way that the origins of individual FN branches are found in the same pattern as the branches are distributed in the face, with the rostral branches represented laterally and the dorsal branches dorsally in the facial nucleus (Fig. 10).

An overlap in representation was seen in the present study between the temporal and the zygomatic branch. Both branches originated from cells located in dorsal regions of the facial nucleus, although with the zygomatic branch the cells of origin were more heavily concentrated on the lateral than the medial aspect of the nucleus, whereas cells giving rise to the temporal branch were more evenly distributed along the medial to lateral axis. The considerable overlap, however, indicates that many cells in the dorsal region of the facial nucleus innervate the muscles in the dorsal head, including the frontalis, although their axons may course as two different branches as they exit the cranium.

Fig. 6. The distribution of labelled cells (indicated by dots) following HRP application to combined stylohyoid and posterior digastric branches. 87: genu of the facial nerve; Mo5: motor trigeminal nucleus; Pr5: principal sensory trigeminal nucleus; Sp50: nucleus of the spinal tract of the trigeminal nerve, oral part; Tr5: spinal tract of the trigeminal nerve; 6: abducens nucleus; 7: facial nucleus; 7N: facial nerve root.

RAT FACIAL MOTOR NERVE

Fig. 7. Labelled facial motoneurons following HRP application to the buccal branch and the upper division of the marginal mandibular branch. Note that labelled cells are of the multipolar type. The section was counterstained with thionin. Scale bar: 100 pm.

TABLE 4. Labelling in the Geniculate Ganglion (GG) Following HRP Application to Peripheral Branches of the Facial Nerve Distal to the Stylomastoid Foramen'

Peripheral branches exposed to HRP % Labelled cells Posterior Marginal Stylohyoid and auricular Temuoral Zvnomatic Buccal mandibular Cervical uosterior dieastric Main GG Dorsal GG Combined GG

X X X X X X X X X X

X X X

X X X X x X X X X X X X

X X X

X X

29.0 22.8 16.1 16.0 14.7 13.2 10.5 9.3 9.3 7.9 1.3 0.8 0.6 0.2 0.1 - - -

29.0 35.5 40.0 6.0 5.7

17.6 7.1

21.4

9.4 -

- -

TL2 - -

0.7

29.0 23.2 17.2 15.4 14.4 13.4 10.2 9.5 9.2 8.0 1.2 0.8 0.6 0.2 0.1 -

- - - n X 0.3 4.3 0.4

X 0.3 - 0.3 X - - -

Control: HRP applied to all branches without transection 0.1 - 0.1

'Each line in the table displays data from an individual rat. Sixteen of the 23 rats were taken from Semba et al. ('84) for comparison and summary. 'TL, tissue was lost.

dGG 'N

GSPN

medial

t c a u c i a l

Fig. 8. A diagram illustrating the geniculate ganglion (GG shaded area) in relation to the intermediate nerve (IN) and the facial nerve (7N) in a top view. The GG consists of the dorsal (dGG) and the main part (mGG). The mGG is an elongated, pyramidal structure, about 1.8 mm in length, extend- ing rostrally from the facial nerve trunk. At the apex of the mGG, the greater superficial petrosal nerve (GSPN) originates. The dGG is much smaller in size and is located medial and dorsal to the mGG, along the IN. Combined, the entire GG contains about 1,000 cells, the longest dimensions of which range from 10 to 50 pm (Semba et al., '84).

Another considerable overlap was seen between the representations of the marginal mandibular branch, particularly its upper division, and the buccal branch. These nerves innervate the nasolabial muscles that control vibrissal movement (see below).

In contrast to the above overlaps, the representation of the posterior auricular branch, mostly in the ventral M subnucleus, and that of the cervical branch, mostly in VM, were rather discrete, each with essentially no overlaps with those of other branches.

The above observations confirmed and extended those reported previously in the rat on the basis of chromatolysis following selective nerve cut (Papez, '27) or antidromic field potentials (Martin and Lodge, '77). In addition, the present findings are in close agreement with some of the more recent observations using intramuscular HRP injections in the rat (Watson et al., '82) and mouse (Komiyama et al., '84), although they are in partial conflict with others (Ash- well, '82; Hinrichsen and Watson, '84).

The organization of the facial nucleus has been studied in a variety of mammalian species other than rodents (Ya- gita, '10; Papez, '27; Vraa-Jensen, '42; Szentagothai, '48; Kitai et al., '72; Dom et al., '73; Provis, '77; Kume et al., '78; Dom, '82; see also Vraa-Jensen, '42; and Courville, '66b, for additional references of early work), and the basic plan of the organization appears to be the same, reflecting the common embryonic development of facial musculature (Huber, '31; Vraa-Jensen, '42: see also discussion by Provis,

Fig. 9. Distribution of labelled cells (dots) in horizontal sections of the dorsal (dGG) and main geniculate ganglion (mGG), following HRP application to the posterior auricular branch. Medial, top; rostral: left. Horizontally shaded areas indicate unlabelled GG cells, whereas vertically shaded areas indicate sensory fibers, which stained darker than motor fibers with thionin. Similar to the observations in Semba et al. ('84), labelled cells are distributed in all regions of the mGG, except for the ventromedial aspect near the facial nerve trunk, where labelling appears somewhat sparse. Labelled cells were of the pseudounipolar type, and generally larger than unlabelled GG cells. GSPN: greater superficial petrosal nerve; I N intermediate nerve; 7N: facial nerve.

\ 230

500 p m


'77; and Watson et al., '82). For example, Vraa-Jensen ('42) found in the dog that all the branches innervating the muscles derived from the embryonic platysma (including the posterior auricular, cervical, and lower marginal mandibular branches) are represented medially, whereas those innervating the muscles derived from the embryonic sphinctor colli profundus (including the temporal, zygomatic, and buccal branches) are represented laterally in the facial nucleus.

However, there are also some variations within this basic plan among different species, which may reflect species- specific development of facial muscles. For example, in the cat, the M subnucleus, which contains motoneurons supplying the auricular muscles, is relatively large, perhaps reflecting the importance of pinna orientation in this species (Papez, '27; Courville, '66b; Kume et al., '78). Similar observations have been made in the brush-tailed possum (Provis, '77). On the other hand, in the North American opossum, the M subnucleus is rather small, while the L subnucleus is large, probably reflecting the importance of defense re- sponse characterized by vibrissal protraction in this species (Dom et al., '73). With the cat and the North American opossum as the two extremes on the scale of relative size of the nasolabial to caudal facial musculature, the rat seems to be close to the North American opossum, with a relatively large L subnucleus.

The suprafacial nucleus. In the present study, we observed that cells contributing fibers to combined stylohyoid and posterior digastric branches are located not in the main facial nucleus, but dorsal to it, plus in the reticular formation between the facial nucleus and the motor trigeminal nucleus. This group of cells is referred to as the suprafacial nucleus or the accessory facial nucleus. In the reticular formation, the location of these cells shifts dorsolaterally in the caudal to rostral direction, with the number of cells becoming fewer toward the caudal tip of the motor trigeminal nucleus. This region of the motor trigeminal nucleus contains cells whose axons innervate the anterior belly of the digastric muscle (Mizuno et al., '75; Jacquin et al., '83).

The above observations agree with those reported in several previous studies in the rat (Mizuno et al., '75; Matsuda et al., '79; Watson et al., '82; Shohara and Sakai, '84) and mouse (Komiyama et al., '84) in which the retrograde HRP transport technique was used. A similar observation has also been made by using the cobalt precipitation technique in the rat (Martin and Mason, '77). Similar observations have been reported in other species as well, including monkey, cat, dog, and guinea pig (Mizuno et al., '75; Matsuda et al., '79). On the other hand, the main facial nucleus has been reported to be the origin or an additional origin of motoneurons supplying the stylohyoid and/or posterior digastric muscle, with the aid of a variety of techniques including the HRP transport technique, in the rat (Martin and Lodge, '77; Hinrichsen and Watson, '841, mouse (Ash- well, '821, dog (Vraa-Jensen, '42), and guinea pig (Papez, '27). In addition, in the cat the stylohyoid muscle has been reported to be supplied by motoneurons located within the facial nucleus, whereas the posterior belly of the digastric supplied by those in the suprafacial nucleus (Szentagothai, '48; Kume et al., '78).

In the reticular formation, between the facial and the motor trigeminal nucleus, Matsuda et al. ('79) observed that labelled posterior digastric cells form a rostral and a caudal group. Similar observations have been made by Szekely and Matesz ('82) following cobalt application to the

B

__-- MAR MAN

P AUR

dorsal

t medial

C

dor' I - m e d i a l

Fig. 10. Summary of the results of retrograde HRP labelling illustrating the relationship between the facial subnuclei and the peripheral facial nerve branches. A. The subnuclei of the facial nucleus, and the suprafacial nucleus. Abbreviations as in Figure 2. B. Representation of the peripheral facial nerve branches in the facial and the suprafacial nuclei (see also Table 3). C. A "rattunculus" in the facial nucleus on the basis of the data summarized in B.


entire FN root. In the present study, however, we observed, in agreement with Shohara and Sakai ('84), that cells labelled following HRP to both stylohyoid and posterior digastric branches became intermittent and sparse at rostra1 levels, but that the separation of labelled cells into two groups was usually not clear.

Afferent fibers in the facial "motor" nerve The present study confirmed our earlier report in the rat

(Semba et al., '84) that up to 30% of the cells in the GG contribute fibers to the posterior auricular branch. This is also in agreement with earlier studies by others in the rat (Martin and Mason, '77; Gomez, '78) and cat (Foley and DuBois, '43; Bruesch, '44). In addition, centrally, at least some of these fibers were seen ipsilaterally to enter the nucleus interpolaris of the spinal tract of the trigeminal nerve, an observation in accordance with those involving the entire FN in the rat (Torvik, '56; Rhoton, '68; Martin and Mason, '77; Contreras et al., '82), cat (Ken-, '62; Rhoton, '68; Arvidsson and Thomander, '84), and monkey (Rhoton, '68).

It has been shown in the cat that some sensory fibers of the vagal origin travel with the FN, to be distributed mostly to the posterior auricular branch (Thomander et al., '82; Arvidsson and Thomander, '84). The present study did not examine this possibility in the rat.

We have previously observed that, in contrast to the posterior auricular branch, the other, more distal branches did not contain a substantial number of afferent fibers originating from GG cells (Semba et al., '84). This observation was confirmed in the present study, and, in addition, we found that neither of the additional two branches of the FN, i.e., the stylohyoid and posterior digastric branches, contained a significant number of such fibers. All of these observations are consistent with previous findings by Martin and Mason ('771, using the cobalt precipitation technique, as well as those by Martin and Biscoe ('77), using synaptic potential recordings in the rat. On the other hand, in the cat some labelled cells were seen in the GG following HRP application to the buccal and the zygomatic branch (Thom- ander et al., '82; see also Foley and DuBois, '43; Bruesch, '44; and Iwata et al., '72), although Boudreau et al. ('71) did not find GG cells that responded to rubbing of the facial muscles.

Some cells in the mesencephalic trigeminal nucleus, the area concerned with the proprioception of jaw muscles, (e.g., Cody et al., '721, have been reported in the mouse to contribute fibers to the facial motor nerve (Fuller, '78). However, in the present study in the rat, none of our HRP applications was followed by labelling in the mesencephalic trigeminal nucleus.

All these findings indicate the paucity of afferent fibers in all the branches of the FN distal to the posterior auricular branch, including those innervating the muscles to produce vibrissal movement. Are these muscles then supplied with sensory fibers of the trigeminal origin, as suggested, for example, by Fuller ('78; see also Brodal., '81)? Examin- ing the levator labii superioris, a major muscle presumably involved in vibrissal movement, Bowden and Mahran ('56) found no muscle spindles, although nerve fiber endings of unknown origin and function were found. In the present study, none of the HRP applications was followed by labelling in the mesencephalic trigeminal nucleus or trigeminal ganglion. All these findings, in combination, suggest that if the muscles controlling vibrissal movement in the rat

contains sensory receptors, they must be innervated by peripheral branches of the trigeminal nerve and these branches must not travel centripetally with the FN.

Control of vibrissal movement by the facial nerve The behavioral testing in the present study indicated that

vibrissal movement in the rat is controlled by two branches of the FN: the buccal branch and the marginal mandibular branch (specifically, its upper division). Retrograde labelling following HRP application to the buccal branch and the upper division of the marginal mandibular branch indicated that the two branches arise from closely overlap- ping regions in the lateral aspect of the facial nucleus, particularly the L and I subnuclei, and that about half of the cells in the facial nucleus contribute fibers to the combined two branches. In addition, examination of anterograde/transganglionic HRP labelling in the GG, brainstem, and spinal cord indicated that these branches contain very few afferent fibers, suggesting that the blockade of vibrissal movement is most likely due to transection of motor rather than sensory fibers.

In the opossum, unlike in the rat, the zygomatic (Dom et al., '73) plus the buccolabial branch (Provis, '77) have been shown to control vibrissal movement. The difference seems to reflect the fact that the opossum has an elongated face, and the nasolabial muscles, controlling vibrissal movement, are innervated not only by the buccolabial branch but also by the zygomatic branch.

Which nasolabial muscles control vibrissal movement in the rat? Dorfl ('82) studied the muscles around the muzzle of the mouse and categorized them into two groups: the extrinsic muscles that are inserted closely into the vibrissal pad but attached to the regions of the skull outside the pad, and the intrinsic muscles that are associated solely with the vibrissal follicles without bone attachment. Although the pattern of innervation of these muscles by the FN branches is not known, Klein and Rhoades ('85) have shown in the rat that all the intrinsic muscles, i.e., those surrounding individual hair follicles, are innervated by cells located in the L subnucleus, and in a somatotopic manner. Similar observations have been reported in the mouse (Komiyama et al., '84). In light of the present findings, these studies suggest that the intrinsic muscles, at least in part, control vibrissal movement, and that the motor fibers innervating them travel through either the buccal or the upper marginal mandibular branch. The role of extrinsic muscles in vibrissal movement, although presumably important, could not be precisely determined in the present study because the nerve-cutting procedure used did not differentiate the innervation of intrinsic and extrinsic muscles.

Whatever muscles actually produce vibrissal movement, the blockade of such movement by cutting the buccal and upper marginal mandibular branches, in combination with the anatomical findings in the present study, indicate that vibrissal movement is controlled by motor fibers originating from cells in the lateral aspect of the facial nucleus. Retrograde HRP labelling indicated that these cells ac- count for as many as about half of the total cell population in the facial nucleus. However, considering the relative volume of the nasolabial musculature (approximately 40% of total facial musculature in the mouse, according to Ash- well, '82), the number of motoneurons involved in vibrissal movement does not seem to be disproportionately large in the rat. Similar conclusions have been arrived at by using intramuscular HXP injections in the rat (Watson et al., '82;


Hinrichsen and Watson, '84; Klein and Rhoades, '84) and mouse (Ashwell, '82; Komiyama et al., '84). As suggested by several authors (e.g., Erzurumlu and Killackey, '79; Watson et al., '82), this may be due to the stereotypic nature of whisking movement of vibrissae (Welker, '64).

Although the density of innervation of muscles controlling vibrissal movement does not seem to be particularly high as compared to that for other facial movements, there seems to be some redundancy in the innervation of the muscles controlling vibrissal movement. That is, cutting either the buccal branch or the upper marginal mandibular branch apparently did not affect vibrissal movement, but both branches needed to be cut in order to block vibrissal movement completely. Although the precise pattern of muscular innervation by the motor fibers contained in the two branches is unknown, this redundancy may represent a built-in safety mechanism for the movement of the vibrissae, a biologically important sensory organ in the rat.

Lateral portions of the facial nucleus, in which, as we have shown, motoneurons evoking vibrissal movement are located, receive preferential projections from several areas of the brain. In an anatomical study, for example, Erzur- umlu and Killackey ('79) have shown in the rat that a selective area of the spinal trigeminal nucleus that contains spatial representation of the vibrissae projects prefer- entially to the lateral aspect of the facial nucleus. Selective af€erents have also been shown to the lateral facial nucleus from the parabrachial nuclear complex, the area implicated in pneumotaxic function (Bertrand and Hugelin, '71) as well as in taste (Norgren and Pfaffman, '75), in the cat (Takeuchi et al., '79, '80), opossum (Panneton and Martin, '831, and rat (Hinrichsen and Watson, '83; Isokawa and Komisaruk, '83; Travers and Norgren, '83). In addition, the lateral facial nucleus receives focussed projections from the nucleus ambiguus, an area containing upper respiratory motoneurons, in the rabbit (Bystrzycka and Nail, '83) and rat (Hinrichsen and Watson, '83; Isokawa and Komisaruk, '83). The red nucleus also projects predominantly to lateral regions of the facial nucleus in the monkey (Miller and Strominger, '73), opossum (Panneton and Martin, '79, '831, rat (Hinrichsen and Watson, '83; Isokawa and Komisaruk, '83; Travers and Norgren, '83), and cat (Courville, '66a; Edwards, '72; Holstege et al., '84). In the rat, such selective projections to the lateral facial nucleus may be important in sensorimotor integration of the vibrissae as a tactile organ as well as in synchronization of vibrissal movement, sniffing, and rhythmical movements of the nose and head during exploratory behavior in this species (Welker, '64).

ACKNOWLEDGMENTS We thank Dr. R.G. Nagele for technical advice regarding

the decalcification of the temporal bone, Dr. G. Macdonald for supplying rats, Ms. S. Harris, Ms. P. Masarachia, Dr. R.W. Rhoades, Dr. N.Y. Shu, Ms. R. Sood, and Ms. V. Sood for technical assistance, and Dr. K. Satoh for critical com- ments on an early version of the manuscript. Support was provided by a General Research Support Grant from Rut- gers Medical School to K.S. and NSF Grant BNS-8205598 to M.D.E.

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The facial “motor” nerve of the rat: Control of vibrissal movement and examination of motor and sensory components