Distribution of motoneurons supplying feline neck muscles taking origin from the shoulder girdle

Distribution of Motoneurons SupplyingFeline Neck Muscles Taking Origin

From the Shoulder Girdle

T.L. LIINAMAA, J. KEANE, AND F.J.R. RICHMOND*

Queen’s University, Kingston, Ontario, Canada, K7L 3N6

ABSTRACTA combination of fluorescent retrograde tracers and horseradish peroxidase (HRP) was

used to compare the spinal distributions of motoneurons supplying shoulder muscles withattachments to the skull and cervical spinal cord that suggest a significant role in headmovement. Two muscles, the rhomboideus and the levator scapulae, were innervated bymultiple segmental nerve bundles that entered the muscles at different rostrocaudallocations. Motoneurons that were labelled retrogradely from rhomboideus nerve bundlesformed a single, long column in the ventral horn from C4 to C6, lateral to previously studiedmotor nuclei supplying deep neck muscles. When different tracers were used to differentiatemotoneurons supplying specific nerve bundles, discrete subnuclei could be identified thatwere organized in a rostrocaudal sequence corresponding to the rostrocaudal order of thenerve bundles. Levator scapulae motoneurons formed a second elongate column immediatelylateral to the rhomboideus motor nucleus. Three other muscles, the trapezius, sternomastoi-deus, and cleidomastoideus, were supplied by cranial nerve XI. Labelled motoneurons fromthese muscles formed a single column from the spinomedullary junction to middle C6. Withinthis column, the three motor nuclei supplying the sternomastoideus, cleidomastoideus, andtrapezius were laminated mediolaterally. Sternomastoideus and cleidomastoideus motoneu-rons were confined to upper cervical segments, whereas trapezius motoneurons were foundfrom C1 to C6. In C1 and C6, the motoneuron column was located centrally in the gray matter,but, between C2 and C5, the column lay on the lateral wall of the ventral horn in a positiondorsolateral to motor nuclei supplying the rhomboideus and the deeper neck muscles. Thefindings in this study suggest that descending and propriospinal systems responsible forcoordinating head movement may have to descend as far caudally as C6 if they are to projectonto muscles controlling the mobility of the lower neck. J. Comp. Neurol. 377:298–312,1997. r 1997 Wiley-Liss, Inc.

Indexing terms: FluoroRuby; fluorescein-conjugated dextran; fast blue; Fluorogold; spinal

accessory nerve

Over the past two decades, much has been written aboutthe distribution of motoneurons that supply muscles sub-serving head movement. However, these studies havefocused almost exclusively on a subset of muscles spanningbetween the skull and the cervical column with motoneu-rons that are located in upper cervical spinal segments.From such results, it has been tempting to conclude thatthe spinal neural machinery responsible for the control ofhead movement is confined largely to segments rostral toC4. This view is reflected further in the protocols ofnumerous electrophysiological and pathway-mapping stud-ies that were undertaken to understand the anatomicalsubstrate of head-movement control and that have gener-ally restricted their analyses to projections ending in the

upper cervical cord (see, e.g., Shinoda et al., 1992; Rose etal., 1992).However, not all neck muscles that participate in head

movement run between the skull and the cervical column.Recent biomechanical and electromyographic (EMG) analy-ses have provided strong evidence that most head move-

Contract grant sponsor: Medical Research Council of Chicago*Correspondence to: F.J.R. Richmond, MRC Group in Sensory-Motor

Neuroscience, Botterell Hall, Queen’s University, Kingston, Ontario K7L3N6, Canada. E-mail: [email protected] 3 April 1996; Revised 13 August 1996; Accepted 6 September

1996

THE JOURNAL OF COMPARATIVE NEUROLOGY 377:298–312 (1997)

r 1997 WILEY-LISS, INC.

ments are produced by joint-angle changes at lower as wellas upper cervical joints (Richmond et al., 1992; Keshner,1994; Graf et al., 1995). Furthermore, these movementsare associated with activity in a diverse mix of muscles,including a number of muscles that cross the lower cervicaljoints from attachments on the shoulder girdle (Keshner etal., 1992; Richmond et al., 1992; Thomson et al., 1994).These more caudally placed muscles with scapular andclavicular attachments are not supplied by segmentalmotor nerves arising from the upper cervical cord (Reighardet al., 1963). Instead, some derive their innervation frompoorly characterized motor nuclei in lower cervical seg-ments. Others are innervated from the spinal accessorynucleus (SAN; cranial nerve XI), a nucleus with internaltopographic relationships that have been controversial.Allinvestigators who have examined the SAN agree that thenucleus contains the motoneurons for the five muscleheads that, together, constitute the trapezius (TRAP) andsternocleidomastoideus muscles. However, some have re-ported that the nucleus is formed by a single column inwhich motoneurons supplying different heads intermingle(Ruminska-Kowalska et al., 1976; Augustine and White,1986; Brichta et al., 1987). Others describe it as a pair ofmediolaterally separated columns in which the motoneu-rons supplying different muscle heads are segregated fromone another (Rapoport, 1978; Krammer et al., 1987) or areintermixed (Kitamura and Sakai, 1982). A better knowl-edge of the intraspinal distribution of motoneurons supply-ing a full range of neck muscles is an important prerequi-site for neuroanatomical and electrophysiological studiesto understand the orchestration of muscles that subservehead movement by acting at different levels of the cervicalvertebral column.In the present study, we have used a range of retrograde

neuroanatomical tracers to examine the distribution ofmotoneurons supplying several neck muscles that origi-nate from bones of the shoulder girdle. Five muscle heads,the clavotrapezius (CT), acromiotrapezius (AT), spinotrape-zius (ST), sternomastoideus (STM), and cleidomastoideus(CLM), form a superficial muscular layer that is suppliedby the spinal accessory nerve. Three other muscles, therhomboideus capitis (RHCAP; also called the occipitoscapu-laris), rhomboideus minor (RHMIN), and levator scapulae(LS), together, form a deeper grouping of muscles thatlinks the scapula to the head and cervical column. Astriking feature of these muscle groupings is their broad,sheet-like architecture in which in-parallel subregions ofmotor units appear to be innervated by different muscle-nerve branches. In other neck and limb muscles, this typeof multiple innervation often predicts the presence ofmultiple ‘‘compartments’’ of motor units (Letbetter, 1974;English and Letbetter, 1982) with motoneurons that mayor may not have different patterns of distribution (forreview, see Gordon et al., 1991). The separation of nervebranches provides an opportunity not only to evaluate thedistribution of motor nuclei as whole entities but also toexamine the relative distributions of motoneurons supply-ing different muscle subregions. Results suggest thatmotoneurons supplying these cranial and axial musclesare not distributed according to similar organizing prin-ciples. Muscles supplied by segmental motor nerves had ahighly ordered pattern of motoneuronal distribution inwhich sequentially arranged subsets of motoneurons sup-plied different muscle-nerve bundles. However, muscles

supplied by the spinal accessory nerve had a much looserpattern of organization in which motoneurons supplyingdifferent muscle-nerve bundles interdigitated extensively.

MATERIALS AND METHODS

Experiments were conducted under aseptic conditionson 20 cats (2.5–4.3 kg) that were anaesthetized withsodium pentobarbital (Somnotol, MTC; initial dose 35mg/kg i.p., supplemental doses 5 mg/kg i.v. to abolishwithdrawal reflexes). An antibiotic (Penlong S, Rogan/ST;0.5 ml i.m.) was administered prior to and after thesurgery. In nine cats, we investigated the distribution ofmotoneurons supplying RH and LS muscles (series 1). Ineight more cats, we examined the distribution of motoneu-rons supplying different heads of the TRAP muscle com-plex (series 2). In the last three cats, we studied therelative distributions of motoneurons supplying the STM,CLM, and TRAP (series 3). Prior to the experiments, fouradditional cat cadavers were dissected to determine themost appropriate surgical approaches.

Series 1: RH and LS

The dorsal midline of the neck was incised between theocciput and dorsal margin of the scapula. The CT wasdissected from its vertebral attachment to gain access tothe underlying RHmuscles. The long nerve bundle supply-ing RH CAP was identified and cut. The scapula wasretracted laterally to expose three shorter bundles ofnerves, which penetrate LS and then cross the cleavageplane between RH and LS to enter RH MIN on its ventralsurface (Fig. 1A). Nerve branches to RH MIN were stimu-lated electrically (0.1 millisecond single pulses, four timesthreshold for just detectable contractions) to confirm thatthey elicited contractions in separate, in-parallel strips ofmuscle around their muscle entry points, as has beenpreviously described (Liinamaa and Richmond, 1994).Nerves supplying LS were more difficult to isolate.

Initially, they share the same course with RH MIN nerveswithin the substance of LS, but they terminate beforereaching its superficial surface. Access to LS nerves couldbe gained only by microdissecting apart the muscle-fiberfascicles at the site of emergence of RHMIN nerve bundlesuntil the multiple fine bundles supplying LS could beidentified within the substance of the muscle (Fig. 1B). Inseven experiments (Table 1), we attempted to label thehidden LS nerve axons by injecting horseradish peroxi-dase (HRP; grade 1; Boehringer) into the muscle or byplacing a tracer-soaked pad of gelfoam over the partiallydissected and sectioned muscle-nerve branches. To labelRH motoneurons in the same experiments, we then coatedthe LS muscle with a thick wax layer through which onlythe nerves supplying RH were allowed to penetrate. Thenerves were cut close to their points of muscle entry, andthe proximal cut ends were laid into reservoirs carved inthe wax (Gordon and Richmond, 1990; Gordon et al., 1991;Kitamura and Richmond, 1994). Each cut nerve wassoaked in distilled water for 10 minutes. The reservoir waschecked for leakage and was then blotted and filled with adrop of tracer solution, as specified in Table 1. In all cases,tracers were dissolved in 0.9% saline to which 2% dimeth-ylsulfoxide was added (Huisman et al., 1982). The filledreservoirs were sealed with a fresh layer of wax, and morewax was spread over the exposed tissues. After 2–3 hours,

NECK MUSCLE MOTONEURONS 299

the wax overlying the reservoirs was removed, and thereservoirs were checked to ensure that no leakage of tracerhad occurred. Tracers were blotted, and the nerves wererinsed with saline solution. The incisions were sutured,and the cats were allowed to recover for 5–7 days.In the early experiments, RH motoneurons were la-

belled effectively by fluorescent tracers, but few, if any,HRP- or fluorescence-labelled motoneurons supplying LScould be identified. Thus, in the later experiments, we useda different method to examine the relative distributions ofLS motoneurons (Table 1). In these cats, the fine nervebranches supplying LS were carefully dissected for about5–10 mm intramuscularly. Care was taken to isolate thelonger and thicker RH nerves from the LS bundles, and the

two sets of nerves were cut as distally as possible. The LSnerve branches and the RH bundles were isolated inseparate wax reservoirs (Fig. 1C) and were exposed totracer solutions like those used to expose RH nerves inearlier experiments (described above).

Series 2: TRAP

An incision was made on the dorsal midline between theocciput and the upper thoracic vertebral column. Themidline attachment of the CT and the AT was cut andretracted to expose the spinal accessory nerve as it coursedcaudally across the ventral surface of the muscle heads. Aseries of branches enter the TRAP at different rostrocau-

Fig. 1. A: Surgical approach to rhomboideus (RH) muscle nerves.The scapula was retracted laterally, and the rhomboideus capitis (RHCAP) and rhomboideus minor (RH MIN) were retracted to expose theRH nerves as they exited from the levator scapulae (LS) to innervateRH. B: A magnification of one set of RH MIN and LS nerves. The

nerves initially travel together as a single nerve bundle as they exitthe spinal cord. When the nerve bundle enters the LS, fine nerves branchoff to innervate the LS. The rest of the nerve bundle exits out of the LS toinnervate the RHMIN. C: The nerves to LS and RH MIN were cut andplaced into separate wax enclosures that contained different tracers.

300 T.L. LIINAMAA ET AL.

dal levels. The CT is generally supplied by the three mostrostral branches, the AT is supplied by two or more middlebranches, and the ST is supplied by the remaining nervebundle, which crosses the caudal edge ofAT (Fig. 2). In fiveexperiments, the distribution patterns of motoneuronssupplying single muscle heads were examined by exposingthe nerve branches that supply a single muscle head to a30% solution of HRP either unilaterally or bilaterally(Table 2). Tracer was applied by isolating the cut nerves inwax reservoirs. In three experiments, a range of fluores-cent tracers was applied sequentially to different indi-vidual nerve branches entering the TRAP (Table 2, Fig. 2).In these latter experiments, care was taken in the initialstages of the nerve dissections to ensure that muscles wereretracted in a manner that permitted the creation of sevenwell-separated and stable reservoirs of tracer. Each nervebranch was stimulated electrically prior to its exposure totracer in order to ascertain the extent of contractionswithin the muscle complex.

Series 3: STM and TRAP

To gain access to nerves supplying ventral muscles of thespinal accessory complex, each cat was laid on its side, andan oblique 6 cm incision was made from the base of the eartowards the forelimb. The border dividing the STM fromthe TRAP was identified, and the muscle heads wereseparated to expose the main nerve bundle, which consti-tuted the spinal accessory nerve, as it coursed caudallyfrom the jugular foramen and gave rise to a series ofbundles supplying the STM, CLM, and TRAP. Each nervebranch was separated until its muscle target becameobvious, was stimulated electrically to confirm this target,

Fig. 2. The three heads of trapezius and their innervation by the spinal accessory nerve. This diagramshows the branching pattern of the spinal accessory nerve. The four fluorescent tracers were exposed in asequential pattern to six or seven cut nerve branches in cats TRZ-1, TRZ-2, and TRZ-3.

TABLE 1. Tracer Application on RH and LS Muscle Nerves1

CAT #RHCAP

RHMIN(R)

RHMIN(M)

RHMIN(C) LS

NOB-1 FR FB FD FG HRP injectionNOB-2 FR FD FG FB HRP-gelfoam caudal

branchNOB-3 FR FB FD FG HRP injectionNOB-4* HRP FG FR FB FD-gelfoamNOB-5 FB FD HRP-gelfoam caudal

branchNOB-6* FD FG FG FB HRP-gelfoamNOB-7 FB FD FG HRP on middle

branchFR on caudal branch

NOB-8 HRP FB FB FB FGNOB-9 HRP FB FB FB FG

*The nerve exposures in NOB-4 and NOB-6 were interrupted during surgery, so thesecats were excluded from the final analysis.1Abbreviations: FB, fast blue; FD, Fluoroscein conjugated Dextran; FG, Fluorogold; FR,Fluoro Ruby; HRP, horseradish peroxidase; LS, levator scapulae; RHCAP, rhomboideuscapitis; RHMIN, rhomboideus minor; r, rostral; m, middle; c, caudal.

TABLE 2. Tracer Application to Spinal Accessory Muscle Nerves1

CAT # CT AT ST STM CLM

THS-10 HRP-allbranches on Rside

ARK-21 HRP-allbranches on Rside



HRP-allbranches on Rand L sides


TRZ-1 Rostral-FB Rostral-FG FDCaudal-FD Middle-FR

Caudal-FBTRZ-2 Rostral-FB Rostral-FR FG

Middle-FD Middle-FBCaudal-FG Caudal-FD

TRZ-3 Rostral-FB Rostral-FR FGMiddle-FD Middle-FBCaudal-FG Caudal-FD

SCM-1 FG FG FG FD FBSCM-2 FG FG FG FB FDSCM-3 FG FG FG FB FR

1Abbreviations: AT, acromiotrapezius; CLM, cleidomastoideus; CT, clavotrapezius; L,left; R, right; ST, spinotrapezius; STM, sternomastoideus and as in Table 1.


and was sectioned. The nerve branches supplying theSTM, CLM, and TRAP were sorted into three collectionsaccording to their targets, and these collections wereexposed to different tracers (Table 2). In some instances,one or more short, rostrally directed branches supplyingthe STM or the CLM proved difficult to isolate; these nervebranches were not exposed to tracer in order to avoidpotential problems that nerves might retract or tracermight leak from unstable wax enclosures.

Perfusion and Histology

Following a 6–8 day survival period (Gordon andRichmond, 1990), cats were deeply reanesthetized withsodium pentobarbital and perfused by delivering fluidunder a controlled driving pressure of 25 kPa through acannula introduced into the descending aorta. Two liters ofnormal saline were perfused followed by 1.5 liters offixative (4% paraformaldehyde in 0.1 M acetate buffer, pH6.5). The cervical spinal cord was removed, and fiduciarymarks were placed at segmental boundaries. In series 3,the medulla was also excised. The tissues were transferredthrough graded concentrations of sucrose in fixative (4%paraformaldehyde in 0.1 M acetate buffer, pH 6.5) over a 3day period. The spinal cord was cut either horizontally orlongitudinally into 50 µm sections. In cats in which HRPwas used, sections were reacted for HRP activity by usingtetramethylbenzidine as the chromogen agent (Mesulam,1978). Sections were mounted onto slides coated withgelatin and were coverslipped with DPX mounting me-dium.The slides were examined by using darkfield and epifluo-

resence illumination. Three filter blocks [Afilter: bandpass340–380 nm, long pass 430 nm; N2.1 filter (Leitz): band-pass 515–560 nm, long pass 580 nm; and I2/3 filter:bandpass 450–490 nm, long pass 515 nm] were used todiscriminate the four fluorescent tracers (Fig. 3). Fluoro-gold (FG; Fluorochrome, Inc.), fast blue (FB; Sigma),and FluoroRuby (FR; Molecular Probes) could be viewedunder the A filter, where they appeared as gold, blue,and pink cells, respectively, as described and illustratedelsewhere (Richmond et al., 1994). When the N2.1 filterwas used, only FR was visible, filling the cell soma,nucleus, and dendrites with a brilliant red granular sub-stance.With the I2/3 filter, only cells containing fluorescein-conjugated dextran (FD; Molecular Probes) and FRwere visible, and these cells appeared yellowish-green andpink, respectively. When using darkfield illumination,HRP was recognized as a dense, copper-colored substancethat filled the cell somata and proximal dendrites. Underfluorescent illumination, HRP-labelled cells appeared asblack profiles.In three cats (NOB-1, NOB-5, and NOB-6), cells in the

contralateral and ipsilateral ventral horn contained accu-mulations of a pale, yellow-colored substance that fluo-resced under both I2/3 andAfilters. Unlike FG, which wasuniformly distributed within the cell soma, this substanceappeared to be clumped nonuniformly in a subregion of thecytoplasm. The material did not have the granularity thatis characteristic of FG but had a diffuse, cloudy appear-ance. Inspection of the contralateral ventral horn con-firmed suspicions that this material was not one of thetracer compounds but an autofluorescing compound that isendogenous to many cells. The presence of the compoundmade it difficult to discriminate with confidence cellslabelled with FG. Thus, the distribution of FG-labelled

cells in these particular cats was not included in thepresent study.The positions of all motoneurons were mapped onto

large drawings by using a camera lucida attachment.Generally, cells were marked onto the drawings if theirnuclei could be seen within the cell somata. However, attimes, some cells were labelled so heavily that the nucleuscould not be seen. Such cells were counted only if the cellsoma and at least two dendrites were visible in the samesection.

RESULTS

Motoneurons supplying RH and LS

Motoneurons supplying RH CAP and RH MIN wereexamined and mapped in six of the nine cats with nervesthat could be exposed to tracer uneventfully for a 2–3 hourperiod (Table 1). In these cats, labelled motoneuronsformed a longitudinal column, which extended from caudalC3 or rostral C4 to middle or caudal C6, that was situatedclose to the ventrolateral margin of the ventral horn (Fig.4). Data from a seventh cat (NOB-1) were excluded fromthe analysis, because FG had been observed to leak fromthe reservoir surrounding an exposed nerve. Subse-quently, many labelled cells were found to be distributedwidely throughout the ventral horns of segments C4–C6 inpatterns suggesting that motoneurons from the underly-ing muscles may have been labelled inadvertently by thetracer. Spinal cords from the remaining two cats (NOB-4and NOB-6) were not included in the analyses, because thenerve exposures were interrupted early in the experi-ments. In one case, the wax exposures were damaged,because the cat coughed. In the other case, excessivebleeding occurred from the cut nerves. Postmortem hema-tological testing revealed that this latter cat suffered fromvonWillebrand’s disease.In all of the six cats on which results were based, nerve

branches were exposed to different tracers to compare thedistributions of their respective motoneurons (Table 1).Motoneurons labelled with different tracers appeared toform largely separate subnuclei that were arranged in arostrocaudal sequence corresponding to the peripheralsequence of the muscle nerves (Fig. 4). The subnucleussupplying RH CAP was located in C4 and had a longitudi-nal length of 7–8mm. In one cat, the rostral limit of the RHCAP nucleus was caudal C3, but, in the other, the nucleuswas contained entirely in C4. The subnuclei for the rostraland middle divisions of RH MIN were located in rostraland caudal C5, respectively. Each had a longitudinallength of 4–5 mm. A small amount of interminglingoccurred at the borders between subnuclei. For example,Figure 4 shows the topographical relationships in one cat,NOB-3, in which six FD-labelled RH MIN (middle) neu-rons were present among the FG-labelled RH MIN (ros-tral) neurons at middle C5. The subnucleus for RH MIN(caudal) was located in C6 and had a rostrocaudal length ofabout 6 mm.The relative locations of LS and RH motoneurons were

compared in three cats in which LS axons were exposed totracer by using the method of cut-nerve exposure (catsNOB-7, NOB-8, and NOB-9). In all of these cats, thelabelled LS motoneurons formed a long column on theventrolateral border of segments C5 and C6. The LS motorcolumn was lateral to that labelled from RH and appeared

NECK MUSCLE MOTONEURONS302

to be sandwiched between the lateral white matter and theRHmotor nucleus. Reconstructions of the two cell columnssuggested that the border between the columns was rela-tively distinct but was not absolute; a small number ofcells labelled with different tracers intermingled along the

line at which the columns appeared to come together (Fig.5). Tracer applied to the caudalmost branches of LSlabelled cells in C6 (cat NOB-7), whereas tracer applied toboth caudal and rostral LS nerves labelled cells in C5 andC6, respectively (cats NOB-8 and NOB-9).

Fig. 3. Histological appearance of labelled motoneurons. A: LSmotoneurons labelled with horseradish peroxidase (HRP) and photo-graphed under combined darkfield and fluorescent illumination (catNOB-2). B: RH motoneurons labelled with fluorescein-conjugateddextran (FD) using the I2/3 filter (cat NOB-2). C: Sternomastoidmotoneuron labelled with FluoroRuby (FR) and photographed usingthe N2.1 filter (cat SCM-3). D: Trapezius motoneurons labelled with

Fluorogold (FG), fast blue (FB), and FR (arrows) and photographedusing the A filter. E: A cluster of trapezius motoneurons labelled withFG and a medially placed sternomastoid cell labelled with FR (catSCM-3). Both were photographed using the A filter. F: A cluster oftrapezius motoneurons (labelled with FG) situated by an adjacentFB-labelled cleidomastoid motoneuron (cat SCM-3). Scale bars 5100 µm.


Motoneurons supplying spinalaccessory musculature

The extent of the motor nucleus supplying the TRAPwas examined in whole or in part in 11 cats by using eitherHRP or multiple fluorescent tracers. In all of these cats,labelledmotoneurons formed a long, narrow column extend-ing fromC1 to middle or caudal C6. The rostralmost pole ofthe motor nucleus, which was found variably from rostralto caudal C1 in different cats, was formed by cells locatedin a central region of the ventral horn (Fig. 6). At morecaudal levels of C1, however, the nucleus shifted laterallyuntil it occupied a site adjacent to the lateral border of theventral horn, where it remained between rostral C2 andcaudal C5. In caudal C5, the motoneuron column shifted

medially again (Fig. 6), so that the caudal pole of thenucleus was positioned midway between the medial andlateral borders of the ventral horn at the level of C6.Motoneurons supplying different muscle heads over-

lapped in their rostrocaudal extents. Motoneurons labelled

Fig. 4. Longitudinal reconstruction (left) and transverse sections(right) of the spinal cord from rostral C4 to caudal C6 showing thedistribution of RH motoneurons in two cats (left, cat NOB-3; right, catNOB-5). In each cat, four tracers were applied to the four nervebranches supplying the RH. Symbols represent RH motoneuronssupplying different nerve branches (black dots, RH CAP; red crosses,RH MIN rostral; black triangles, RH MIN middle; black squares, RHMIN caudal). The three transverse sections each show the distributionof cells over a rostrocaudal extent of 1 mm.

Fig. 5. Diagrammatic reconstruction of a portion of the RH and LSmotor columns, frommiddle C5 to rostral C6 (cat NOB-8). Labelled LSmotoneurons (circles) were situated in a column in the ventrolateralregion of the ventral horn, immediately lateral to the RH motornucleus (squares).


by exposure of CT nerves formed an uninterrupted columnthat usually had its rostral limit in C1 and its caudal limitin C4. In three cats, however, the CT column extended intorostral C5. The cell column labelled from nerves supplyingthe AT was located from rostral C3 to middle C5, and thatlabelled by the ST ran from middle or caudal C3 to middleor caudal C6. The extensively overlapping nature of thesecolumns was demonstrated convincingly in experiments inwhich nerve branches supplying different parts of theTRAPwere labelled with different tracers in the same cats.Neurons labelled with different tracers were found tointermingle extensively between C2 and C5 (Fig. 7). Nodistinct borders or gradients could be identified thatcorresponded to the ordered rostrocaudal sequences oftracers applied as shown in Figure 2. None of the labelledcells contained more than one tracer.STM motoneurons were distributed in a short column

extending from the spinomedullary junction to caudal C1or rostral C2 (Fig. 8). At its rostral pole, the column waslocated near the dorsomedial border of the ventral horn,but it shifted centrally at more caudal levels. CLM moto-neurons were observed to form a column from caudal C1 orrostral C2 to middle or caudal C3. In one of the three catsstudied, a few CLM motoneurons were found in thespinomedullary region and in rostral C1.When different tracers were used to label motor nuclei

supplying the STM, CLM, and TRAP in the same cat, alaminated organization of the three motor columns wasrevealed (Figs. 8, 9). STMmotoneurons formed themedial-most column, which laid alongside the rostral pole of theCLM motor nucleus in caudal C1 and rostral C2. TRAPcells formed the lateralmost column, adjacent to the CLMmotoneurons (Fig. 9). A small degree of interminglingoccurred between cells on the approximated edges of thenuclei. To examine the relative distributions of the threenuclei more quantitatively, the positions of STM, CLM,and TRAP motoneurons in 7 mm of sections from caudalC1 (cat SCM-3) were defined by measuring their dorsoven-tral and mediolateral distances from horizontal and verti-cal axes intersecting at the midpoint of the central canal.An F-value test was used to determine whether the meanmediolateral or dorsoventral positions of the three cellpopulations were located at different sites relative to thecentral canal. Themeanmediolateral positions were foundto differ statistically (P , 0.001); mean dorsoventralpositions did not differ using this criterion.

DISCUSSION

Technical considerations

The recent development of novel retrograde tracers hasrevolutionized the study of neuronal distribution. By label-ling several cell populations concurrently using differenttracers, it is possible to distinguish between closely associ-

Fig. 6. Line drawings of transverse sections of the ventral graymatter to show the distribution of trapezius (TRAP)motoneurons frommiddle C1 to C6. The top four profiles are each compiled from tenconsecutive sections in the rostral cervical cord (cat SCM-1). Thebottom two profiles are both compiled from 15 consecutive sections inthe caudal cervical cord (cat SCM-3). Motoneurons were locatedcentrally within the ventral horn in the rostralmost and caudalmostregions of the nucleus. Between C2 and C5, TRAP motoneurons weresituated adjacent to the lateral border of the ventral horn. r, Rostral;m, middle; c, caudal.

305T.L. LIINAMAA ET AL.

ated sets of neurons, such as those composing the RH andLS motor nuclei or the SAN studied here. However,multilabellingmethods have practical limitations, becausethe nerves under study must be located in multiple,simultaneously accessible sites. This restriction can dic-tate the choice of nerve branches to be exposed in a singlecat. In the present study, for example, the tissue retractionthat was required to ensure the stability of tracer wellsaround the short RH MIN and LS nerve branches pre-cluded further manipulations to expose the deep, rela-tively inaccessible nerves supplying RH major. This delib-

Fig. 7. Diagrammatic reconstruction of rostral C3 in the horizontalplane (cat TRZ-3). The seven branches of the spinal accessory nerve wereexposed in an ordered pattern (see Fig. 2) to four different fluorescenttracers (indicated by different symbols). The subnuclei supplying each ofthese nerve branches intermingled extensively. Abbreviations as in Fig-ure 2.

Fig. 8. Line drawings of transverse sections of the ventral graymatter showing the rostral part of the SAN (cat SCM-1). Each ventralhorn profile was compiled from ten consecutive sections. Labelledsternomastoid motoneurons (circles) formed a short column from thecaudalmost part of the spinomedullary junction (s-m) to caudal C1.These were laminated alongside the cleidomastoid motoneurons(crosses), which, in turn, were positioned lateral to the sternomastoidmotoneurons and medial to the trapezius motoneurons (squares). r,Rostral; m, middle; c, caudal.

NECK MUSCLE MOTONEURONS306

erate omission must be borne in mind when interpretingthe present results, which cannot demonstrate the caudalextent of the RH motor nucleus but, rather, demonstratethe extent of motoneurons supplying heads with cranialand cervical attachments only. Furthermore, it was notpossible to include simultaneous nerve exposures of thedeeply placed muscle, LS ventralis. The location of this

motor nucleus has been described by Hentona (1993) (Fig.10).Nerves can be exposed to some fluorescent tracers by

using either intramuscular injection or cut-nerve expo-sure. In the present studies, intramuscular injections wereattempted first to demonstrate LS motoneurons, but nolabelled cells could be recognized following this procedure.The success of intramuscular injection depends on a finebalance between two requirements. Sufficient tracer mustbe injected to expose all nerves, but too liberal an injectionof tracer will result in leakage and contamination ofadjacent musculature (Richmond et al., 1978; Karim andNah, 1981; Haase andHrycyshyn, 1986; Krogh and Towns,1986). In the present studies, we used small tracer injec-tions, because LS is a thin, sheet-like muscle that isbounded by other muscles, which could easily take up thetracer. Thus, the absence of labelling in the early experi-ments is most likely due to an insufficient delivery oftracer. In contrast, placement of cut nerve branches inwells of tracer was found to produce consistent, well-controlled labelling. Spurious results were rare and oc-curred only when tracer could be seen to leak from adamaged wax enclosure.In the present studies, no specific counts of labelled cells

have been reported because of concerns that such countswould underestimate the size of motoneuron populations.Previous studies have suggested that, for reasons that arepoorly understood, some of the tracers used here (e.g., FB,FR) often may not label all of the motoneurons with axonsthat are exposed to them (cf. Richmond et al., 1994). Thismay reflect, at least in part, the failure of uptake ortransport mechanisms in some nerve axons (Swett et al.,1986). Furthermore, reliable counts will be obtained only ifall muscle-nerve branches supplying a target muscle canbe exposed adequately to tracer. We are reasonably confi-dent that all of the nerves supplying the RH and the TRAPwere exposed to tracer, but the same case cannot be madefor the other muscles, particularly for the LS. The LSnerves are tiny, deeply situated, and numerous. Thus, itonly proved possible to expose a subset of nerve branchesto obtain an approximate picture of the intraspinal relation-ships between the LS motoneurons and the RH motoneu-rons.

Location and extent of motor columnssupplying shoulder muscles

RHand LS. Motor nuclei supplying the RH and the LShave been subjected to little previous study. We know ofonly one previous report in cats, which identified LSmotoneurons in segments C3 and C4 (Hentona, 1993).This pattern of distribution was not confirmed in thepresent studies; instead, RH and LS motoneurons werefound to be distributed more caudally in segments C4–C6(Fig. 4). The more caudal distribution agrees more closelywith distributions of rodent RH motoneurons, which havebeen described to extend as far caudally as C5 (Kitamuraet al., 1980). The presence of RH and LS motoneuronsbetween C4 and C6 has important implications for re-search concerned with the spinal circuitry controlling headmovement. Recent EMG recordings in cats have shownthat RH and LS motoneurons are activated phasicallyduring lifting and turning movements of the head (Thom-son et al., 1994). These patterns of activation presumablydepend on signals carried by descending and propriospinalcircuits that project into caudal parts of the cervical spinal

Fig. 9. Line drawings of the C3 ventral horn comparing thedistributions of TRAP and cleidomastoid (CLM) motoneurons (catSCM-3). TRAPmotoneurons (circles) were situated in a circumscribedregion beside the lateral border of gray matter, immediately adjacentto the CLMmotoneurons (squares).


Fig. 10. Distribution of certain known motor nuclei in C1, C4, andC6. The borders of all nuclei are approximate. The motoneuron nucleiidentified in the present study are compared with the motor nucleiidentified in previous studies on the cervical cord. Distributions of

motor nuclei of deep neck muscles in C1 come from Kitamura andRichmond (1994). Descriptions of motor nuclei in C4 and C6 are basedon drawings from Sterling andKuypers (1967), Richmond et al. (1978),and Hentona (1993).


cord. However, most previous studies of pathways pre-sumed to subserve head movement have concentratedalmost exclusively on projections to upper cervical seg-ments (see, e.g., Rose et al., 1992; Shinoda et al., 1992).SAN. Motoneurons supplying the feline spinal acces-

sory nerve were found to form an elongate column thatextended as far caudally as C6 (Fig. 6). This observation isconsistent with previous reports by Satomi et al. (1985)and Vanner and Rose (1984), who found labelled cells inC6, but is not consistent with reports by other investiga-tors (e.g., Holomanova et al., 1972; Rapoport, 1978), whodescribed a shorter motor column ending in C5. The caudallimit of C6 rather than C5 is consistent with descriptionsof the rodent SAN, in which labelled cells were reported asfar caudally as C6 (Kitamura and Sakai, 1982; Matesz andSzekely, 1983) or even C7 (Krammer et al., 1987; Charltonet al., 1988) following tracer injections into muscles sup-plied by the spinal accessory nerve.Within the SAN, the TRAP motor column had the

longest rostrocaudal extent throughout all but the rostral-most fewmillimeters of the nucleus.Motor columns supply-ing the STM and CLM were confined between the spino-medullary junction and caudal C3 (Figs. 8, 9), as reportedpreviously by Holomanova et al. (1973) and Satomi et al.(1985), who exposed nerves supplying both the STM andthe CLM to a single tracer. The extent of the STM andCLM column was longer than that reported by Rapoport(1978), who used intramuscular injection to label motoneu-rons of the STM/CLM complex and found cells only in C1and rostral C2. The restricted distribution of cells labelledby Rapoport (1978) might be explained if tracer injectionswere confined primarily to STM, which is easier to ap-proach surgically than the deeper CLM. In support of thisinterpretation, STM motoneurons, as differentiated fromCLM motoneurons in this study, were found to be locatedin C1 and rostral C2 in a pattern like that reported byRapoport (1978).Most motor columns in the spinal cord are characterized

by a particular topographic relationship with respect tothe gray-white borders of the ventral horn. The SAN is anunusual motor nucleus, because its mediolateral positionwithin the ventral horn varies according to cervical level(Fig. 6). The present studies confirmed previous findings(Satomi et al., 1985; Augustine and White, 1986; Ueyamaet al., 1990) that the SAN is located quite medially in theC1 ventral horn but gradually adopts a more lateralposition against the lateral gray-white border in cervicalsegments C2–C5 (see Fig. 6). Caudal to C5, the SAN wasagain found in a more medial position, just lateral to thephrenic nucleus (Webber et al., 1979; Rikard-Bell andBystrzycka, 1980; Kitamura and Sakai, 1982; Vanner andRose, 1984; Satomi et al., 1985; Gordon and Richmond,1990). This medial placement of the caudal SAN had notbeen recognized by many previous investigators, presum-ably, because labelled cells could not be identified as farcaudally as C6 (see, e.g., Rexed, 1954; Holomanova et al.,1972; Rapoport, 1978). The failure to identify motoneuronsin C6 in those experiments in which tracer was injectedmay have resulted frommethodological problems in achiev-ing sufficient exposure of nerve branches supplying thecaudalmost muscle head, the ST.Laminar organization of the SAN. Perhaps the sub-

ject of most controversy in studies of the SAN is theorganization of SAN motoneurons into one or more sepa-rate motor columns. Some investigators have described

the SAN as a single column (dog: Ruminska-Kowalska etal., 1976; baboon: Augustine and White, 1986; rabbit:Ullah and Salman, 1986; rat: Brichta et al., 1987), butothers identify two mediolaterally separated columns (rat:Kitamura and Sakai, 1982; Krammer et al., 1987; cat:Rapoport, 1978). Furthermore, the reports suggest differ-ing degrees of muscular segregation within the columns.For example, Rapoport (1978) reported that sternocleido-mastoid motoneurons were confined to a medial column,whereas TRAP motoneurons were found in a lateralcolumn. However, others found CLM motoneurons in boththe medial and the lateral columns (Brichta et al., 1987;Krammer et al., 1987).The present observations based on multiple tracers may

help to resolve the contradictory findings of previous work.By labelling cells of different muscles using separatetracers, TRAP, CLM, and STMmotoneurons were found tobe nested against one another in a laminar pattern (Figs.8, 9). This pattern was recognized most easily in C1 andC2, where all three motor nuclei were represented, withSTM motoneurons the most medial and TRAP motoneu-rons the most lateral. Perhaps it is not surprising thatprevious investigators could not identify this laminarpattern. It is very difficult to discern relationships betweenmotor nuclei when only one tracer is used, because therelative distributions of motoneurons labelled from one oranother muscle must be discerned by comparing resultsfrom different animals. Differences in topography can beidentified with certainty under such conditions only if thecells occupy substantially different intraspinal locations.Topographical relationships can be clouded further ifmotoneurons are not labelled uniformly by the methods ofnerve exposure. For example, if the STM, but not the CLM,motor nucleus was to be labelled by an intramuscularinjection into the sternocleidomastoid complex, then aregion of unlabelled cells (corresponding to unlabelledCLM motoneurons) might appear to separate the TRAPmotor column from the presumptive STM/CLM column (assuggested, e.g., by Rapoport, 1978).A distinct laminar or topographic organization of moto-

neurons supplying different muscles is not unique to theSAN. It has also been observed in the fifth cranial nucleus,which contains the motoneurons of different jaw muscles(Batini et al., 1976; Terashima et al., 1994), in the oculomo-tor nuclei (Porter et al., 1983), in the facial nucleus(Courville, 1966; Dom, 1982; Hinrichsen and Watson,1984), and in the hypoglossal nucleus, which suppliesdifferent muscles of the tongue (Krammer et al., 1979).When considering the functional significance of such closelamination, it is important to remember that the dendritictrees attached to the cell bodies of SAN motoneuronsbranch widely throughout much of the ventral horn(Vanner and Rose, 1984). Whether these dendrites areintermingled randomly or are segregated according to anas yet unidentified plan remains to be determined by othermethods.Subnuclear organization in neckmotor nuclei. Mul-

tiple labellingmethods can also be used to gain insight intothe organizational features of motor nuclei supplyingdifferent parts of complex or compartmentalized muscles.However, in the limited studies that have been conductedto date, no single set of rules has emerged to explain thepatterns of organization that are typical for a compartmen-talized muscle. In previous experiments at two differentlevels of the spinal cord, the lumbosacral and cervical


cords, evaluations of subnuclear organization suggestedquite different organizing principles. In lumbosacral motornuclei, motoneurons supplying different muscle parts weretypically intermixed extensively (e.g., sartorius and tensorfasciae latae: Gordon and Richmond, 1991; semitendino-sus: Letbetter and English, 1981). Extensive intermixingof motoneurons was characteristic even for muscles suchas sartorius that contain two or more compartments ofmotor units with different mechanical actions and differ-ent patterns of EMG activity during movement (Chanaudet al., 1991).In contrast, motor nuclei in cervical segments (including

the RH and LS motor nuclei studied here) have a morestructured organization in which multiple subsets of moto-neurons supplying different muscle regions are arrangedin separate, serially ordered subnuclei (Richmond et al.,1978; Abrahams and Keane, 1984; Armstrong et al., 1988;Gordon and Richmond, 1991). The boundaries betweenthese subnuclei generally coincide with intersegmentalboundaries, but interesting exceptions exist. In biventercervicis, for example, two subsets of motoneurons supply-ing different nerve bundles are known to occupy the rostraland caudal parts of the C3 segment, respectively. Simi-larly, in the RH motor nucleus, two sequentially arrangedsubnuclei were found to be present in rostral and caudalC5, respectively (Fig. 4). Why should two separate subnu-clei be arranged in series within the same spinal segment?It seems to contradict the intuition that the division intosubnuclei results from embryological arrangements inwhich a single spinal segment supplies a single musclemyomere. The observations might be explained if theseven-boned mammalian cervical column could be shownto evolve from species with additional cervical vertebraethat were lost or were condensed over time without aparallel disappearance of the associated neural substruc-ture.The motoneuronal distribution of the spinal accessory

motor nucleus was found to be less regimented than that ofsegmental motor nuclei. Even though the TRAP, like theRH, is supplied by several nerve branches innervatingdifferent muscle heads or parts of heads, extensive inter-digitation was found to occur between motor subpopula-tions supplying different heads (Fig. 7). Patterns of organi-zation weremore similar to those of motor nuclei supplyinghindlimb muscles or, perhaps more notably, the phrenicmotor nucleus against which it abuts in C6 (Webber at al.,1979; Rikard-Bell and Bystrzycka, 1980; Gordon andRichmond, 1990). The observation of such an interdigi-tated organization is particularly intriguing, because thethree muscle heads comprising the TRAP complex arehighly differentiated anatomically and functionally. Thus,it would appear that the cervical origin of a motor nucleusis not enough to guarantee that its motoneurons will bearranged in an orderly pattern. Instead, highly orderedmotor subnuclei appear to be characteristic only for seg-mentally innervated muscles with multiple nerve bundlesthat do not come together in large common peripheraltrunks as they approach their target muscles. Such segre-gated arrangements are generally possible only for axialmuscles with motor axons that have a relatively shortdistance to travel from the spinal cord.

Topography in the cervical spinal cord

The results presented here add to an increasingly com-prehensive picture of motoneuronal distribution in the

cervical spinal cord. In Figure 10, interrelationships arediagrammed between shoulder muscle motoneurons andsome of the motor nuclei with distributions that have beenreported previously. Motoneurons supplying the shouldergirdle tend to lie laterally with respect to other neckmuscles in all but C1 and C6, where the SAN has adistinctive, dorsomedial location. In C1, the dorsomedialpositioning of SCM motoneurons brings them close to themotor columns that lie on the medial wall of the C1 ventralhorn (Kitamura and Richmond, 1994). More caudally,however, TRAPmotoneurons are located laterally near themotor nuclei of muscles, such as splenius (Richmond et al.,1978), that are implicated in head turning (Keshner et al.,1992; Richmond et al., 1992). The proximity to other headturners seems appropriate functionally, because the CT isa fast muscle that is known to be active during movementsaway from the midsagittal plane (Richmond et al., 1992).Of the shoulder muscles studied here, RH motoneurons,which tend to be active during head extension (Richmondet al., 1992), are closest to the base of the ventral horn nearmotoneurons supplying other extensors, such as biventercervicis (Richmond et al., 1978).The patterning of neck and shoulder motor nuclei sug-

gests a rough functional map in which muscles withactions in the sagittal plane are located medially, whereasmuscles acting out of this plane are distributed morelaterally. Previous investigators have suggested that me-diolateral differences in the placement of motor nuclei maybe related to differences in connectivity from differentdescending systems (see, e.g., Brichta et al., 1987). Theirsuggestions would seem to be reinforced by pathway-tracing studies showing that functionally distinct systemsdescend in different parts of the white matter. At the levelof the cervical cord, for example, medial vestibulospinalaxons usually descend in the medial part of the ventralfuniculus (but there are exceptions; see, e.g., Donevan etal., 1992b), lateral vestibulospinal axons and axons fromthe Fields of Forel are common in the ventral part of theventral funiculus, and rubrospinal axons descend in thelateral funiculus (for detailed review, see Holstege, 1988).However, hypotheses that relate motoneuronal location toconnectivity often depend on two assumptions: first, thatthe cell body is the most important part of the neuron forthe receipt of input; and, second, that descending path-ways terminate preferentially on cells that lie closest totheir paths. Both of these assumptions must be consideredcritically.First, it now seems clear that any consideration of

motoneuronal connectivity that focuses on themotoneuronsoma as the primary target may be over-simplistic. Intra-cellular injections of tracer into neck motoneurons havedemonstrated impressive arbors of dendrites that consti-tute as much as 99% of the neuronal surface area (Rose etal., 1985). Frommany motoneurons, dendrites course bothdorsomedially and dorsolaterally along the walls of theventral horn, where they would seem to be accessible to avariety of inputs. Interestingly, TRAP motoneurons seemto defy this pattern:Most TRAPdendrites run in rostrocau-dally directed bundles along the lateral margin of theventral horn and are confined to the motor nucleus (Van-ner and Rose, 1984). Until we understand how dendriticand somatic inputs are organized, it will be difficult toappreciate the role of location of the postsynaptic ele-ments.


Asecond reason for caution when relating motoneuronallocation to connectivity stems from reconstructions ofstained collaterals from descending projections. Thesecollaterals often distribute their contacts widely through-out the ventral and even the dorsal horn. For example,single collaterals of medial vestibulospinal axons havebeen shown to ramify widely in both the ventromedial andspinal accessory nuclei as well as in more dorsal laminae(Donevan et al., 1992a; Shinoda et al., 1992; Rose et al.,1996).Perhaps it is unwise to propose any specific functional

rationale to explain the motoneuronal distributions re-ported here and elsewhere (for further discussion, seeKitamura and Richmond, 1994). The identification ofmotoneuronal location, however, is a significant first stepin the analysis of connectivity. We have now shown thatmuscles known to be active during head movement aresupplied by motoneurons as far caudally as C6. So far, thismidcervical region has been largely neglected by thoseinterested in the neural organization of the spinal cord.Investigators studying head movement have concentratedon upper cervical regions, whereas investigators inter-ested in forelimb control have studied motoneurons caudalto C6. The middle cervical region may prove to be unique,because many of the muscles in this region appear to havea role both in head movement (Richmond and Vidal, 1988)and in forelimb movement (Rushmer et al., 1983). A dualrole in head and forelimb movement suggests a compli-cated array of connections from the two motor systems,which must be arranged precisely if purposeful behaviorsare to be carried out.

ACKNOWLEDGMENTS

We thank Janet Creasy for her technical assistance andSharonWong for her help with illustrations.We also thankDrs. Vivian Abrahams and Ken Rose for their editorialsuggestions and Robin Ashcroft for her help with the finalrevisions. This work was funded by the Medical ResearchCouncil of Canada.

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Documents

Distribution of motoneurons supplying feline neck muscles taking origin from the shoulder girdle