Heterogeneous Projections of the CatPosteroventral Cochlear Nucleus

ANN M. THOMPSON*Department of Otorhinolaryngology, The University of Oklahoma Health Sciences Center,

Oklahoma City, Oklahoma 73190

ABSTRACTThe anterograde tracer Phaseolus vulgaris-leucoagglutinin was used to identify the

projections of the posteroventral cochlear nucleus in cats. After labeling predominately cells ofthe core and multipolar regions, varicose fibers were observed in a variety of auditory nuclei.Ipsilaterally, most varicose fibers were located in periolivary regions situated lateral to themedial superior olive of the superior olivary complex. Contralaterally, the majority of labeledfibers were located in the ventral nucleus of the trapezoid body and the ventral nucleus of thelateral lemniscus. Labeled varicose fibers were also observed in regions not commonlyidentified as receiving input from the posteroventral cochlear nucleus. These regions includedbilaterally the principal nuclei of the superior olivary complex, some periolivary regions, andthe sagulum, as well as the ipsilateral intermediate and dorsal nucleus of the laterallemniscus, inferior colliculus, and lateral pontine nucleus. Both similarities and differenceswere observed in the projections of the core and multipolar regions. With the exception ofcalyceal-type endings in the contralateral ventral nucleus of the lateral lemniscus, thevaricose fibers in all regions, including the contralateral medial nucleus of the trapezoid body,were beaded, en passant type terminal varicosities. J. Comp. Neurol. 390:439–453, 1998.r 1998 Wiley-Liss, Inc.

Indexing terms: auditory pathways; superior olivary complex; inferior colliculus; anterograde

transport

The posterior division of the ventral cochlear nucleus,PVCN, projects to periolivary regions of the superiorolivary complex (SOC) where it terminates onto olivo-cochlear neurons (Robertson and Winter, 1988; Thompsonand Thompson, 1991a; Warr, 1969). Because olivocochlearneurons form descending projections to the cochlea, as wellas collaterals to the cochlear nucleus (Ryan et al., 1990;Brown, 1993), PVCN is thought to have a role in theolivocochlear feedback system (Warr, 1992). Recent stud-ies of PVCN efferent projections to both nonauditory andauditory brainstem nuclei suggest that PVCN has otherroles as well. For example, PVCN has been shown toproject to the lateral paragigantocellularis nucleus, and itsterminations onto serotoninergic and adrenergic neuronsin that nucleus imply that PVCN provides auditory inputto ultimately influence autonomic and limbic function(Kandler and Herbert, 1991; Bellintani-Guardia et al.,1996). Furthermore, PVCN projects to primary nuclei ofthe SOC, including the ipsilateral lateral superior oliveand the contralateral medial nucleus of the trapezoid body(Thompson and Thompson, 1987; 1991b; Friauf and Ost-wald, 1988). Although the postsynaptic cells of the primarySOC nuclei receiving input from PVCN have not beenidentified, their location within the SOC suggests an

additional function of PVCN that is nonolivocochlear andmay be related to the primary ascending auditory path-way.

To better understand the roles of PVCN, a recent goal ofour laboratory has been to elucidate its projections in thecat, using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). Introduced by Gerfen and Saw-chenko (1984), PHA-L has a number of advantages overother tracers. PHA-L is transported preferentially in theanterograde direction and PHA-L-labeled axons and termi-nals can be visualized in detail. Furthermore, iontopho-retic delivery of PHA-L results in small and circumscribedinjection sites so that projections of discrete cellular re-gions within a nucleus can be visualized. The currentstudy reports the results of injecting PHA-L into both thecore and multipolar cell areas of the cat PVCN.

Grant sponsor: National Institutes of Health; Grant number: DC 01058.*Correspondence to: Ann M. Thompson, Ph.D., Department of Otorhino-

laryngology, The University of Oklahoma Health Sciences Center, P.O. Box26901, Oklahoma City, OK 73190. E-mail: ann-thompson@ouhsc.edu

Received 27 June 1996; Revised 20 August 1997; Accepted 22 August1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 390:439–453 (1998)

r 1998 WILEY-LISS, INC.

MATERIALS AND METHODS

Data from 16 adult female cats, in which PHA-L wasinjected unilaterally into both the core (octopus) and/ormultipolar regions of PVCN, are presented. The guidelinesof the Society for Neuroscience regarding the experimentaluse of animals were followed, and all protocols wereapproved by the Institutional Animal Care and Use Com-mittee. For the operations, the cats were anesthetized withan intramuscular injection of a mixture of ketaminehydrochloride (Ketaset, 20 mg/kg) and xylazine (Rompun,3 mg/kg) and maintained with ketamine. The cat’s headwas placed in a stereotaxic frame (David Kopf Instru-ments, Tujunga, CA) to immobilize it during the surgery,and the frame was rotated so that the nose pointed down atan angle of about 50° from horizontal to gain better accessto the cochlear nucleus. The right cochlear nucleus wasapproached through the posterior fossa by first exposingthe midline base of skull. After enlarging the foramenmagnum dorsally and laterally, the dura was slit, and thecerebellum was retracted medially by using saline-saturated wedges of Cottonoid (Codman & Shurtleff, Ran-dolph, MA). The choroid plexus overlying the cochlearnucleus was then retracted upward to visualize the co-chlear nucleus, and a glass micropipette (tip diameter12–13 µm) containing PHA-L was lowered into the PVCNposteriorly to depths of 0.5–1.0 mm. One injection ofPHA-L was made in 15 cats, whereas one cat (cat 8439)received one injection in each of three separate penetra-tions. After injection of PHA-L, the pipette was removed,the craniotomy covered with Gelfilm (Upjohn, Kalamazoo,MI), and the wound closed with suture.

Our laboratory’s basic protocol for PHA-L injection,perfusion, immunohistochemistry, and histology were fol-lowed with volumes increased for the cat (Thompson andThompson, 1987). The cats were overdosed 8–10 days afterthe PHA-L injection with sodium pentobarbital (150 mg/kg, i.p.) and perfused intracardially. The brains weresectioned with a sliding microtome from the obex throughthe inferior colliculus at 40-µm thickness. In all animals,the PHA-L was visualized with the avidin biotin complextechnique (ABCkit, Vector Labs, Burlingame, CA) withdiaminobenzidine dihydrochloride (DAB) as the chromo-gen (Hsu et al., 1981). The DAB in alternate sections ofthree animals was intensified with nickel ammoniumsulfate. After processing, all sections were serialized andmounted onto slides and then cleared and coverslipped.

Some sections, including those through the cochlearnucleus, were counterstained with cresyl violet.

The sections were viewed with a Nikon Optiphot-2 lightmicroscope; a drawing tube attachment was used to makereconstructions. To reconstruct the injection sites, thelocation of labeled cell bodies in the injected cochlearnucleus was drawn under a 34 objective in every fourthsection. A cell body was considered labeled if it containedbrown staining typical of the DAB reaction product. Inregions where background labeling was present, a cell wasconsidered labeled if it was distinguishable from thebackground by a more intense color and if its dendrites oraxon were also visible. Areas of dense background labelingthat would obscure any labeled cell body were also drawnas part of the injection site. Because granule cells areknown to supply only intrinsic cochlear nucleus projec-tions, they were excluded from the reconstructions (Braweret al., 1974; Mugnaini et al., 1980; Oertel and Wu, 1989).

Reconstructions of the injection sites were transferred toa PVCN template that was made from sections of one cat(1028), by utilizing previous works (Osen, 1969a; Braweret al., 1974; Liberman, 1993), as well as landmarks andboundaries that were distinguishable in all injected ani-mals. The PVCN was, therefore, divided into a core region(octopus cell region), a multipolar cell region, and ananterodorsal division. The small cell cap and granule celllayer surrounded these areas.

The sections were viewed with 320 and 340 objectivesto find and trace PHA-L-labeled axons and varicose fibers,which were ultimately mapped by using a 310 objective. Avaricosity was defined as a swelling that occurred along afiber segment, with the segment being at least the lengthof the diameter of the associated varicosity. Each nucleusor region that contained a varicose fiber, regardless of howmany, was noted. For each SOC, independent of the SOCon the other side, the relative numbers of varicositieswithin each nucleus and region were assessed under a 340objective by using a subjective ordinal scale. For the scale,a 1 value was given if varicosities were sparse and a 11value was noted if varicosities were more readily observ-able but were found in only one to a few sections. Ifvaricosities were present in most sections and if they werereadily observable with a 310 objective, then a value of111 was given. Drawings of labeled fibers and varicosi-ties were made with the drawing tube by using a 340objective. The parcellation and nomenclature of the SOC

AD anterodorsal region of posteroventral cochlear nucleusALPO anterolateral periolivary nucleusAVCN anteroventral cochlear nucleusBP brachium pontisC core region of posteroventral cochlear nucleus (octopus cell

area)DAS dorsal acoustic striaDCN dorsal cochlear nucleusDLPO dorsolateral periolivary nucleusDMPO dorsomedial periolivary nucleusDNLL dorsal nucleus of the lateral lemniscusIAS intermediate acoustic striaIC inferior colliculusINLL intermediate nucleus of the lateral lemniscusLNTB lateral nucleus of the trapezoid bodyLSO lateral superior oliveLTTB lateral tract of the trapezoid bodyLPN lateral pontine nucleus

M multipolar region of posteroventral cochlear nucleusMDPO mediodorsal periolivary nucleusML medial lemniscusMNTB medial nucleus of the trapezoid bodyMSO medial superior oliveP pyramidal tractPHA-L Phaseolus vulgaris-leucoagglutininPPO posterior periolivary nucleusPV posteroventral region of anteroventral cochlear nucleusPVCN posteroventral cochlear nucleusRB restiform bodySag sagulumSOC superior olivary complexVII facial motor nucleusVNLL ventral nucleus of the lateral lemniscusVNTB ventral nucleus of the trapezoid bodyVst spinal trigeminal tract

Abbreviations

440 A.M. THOMPSON

followed that of Spirou and Berrebi (1996) with theaddition of the mediodorsal periolivary nucleus, which islocated in the dorsal SOC, between the medial and lateralsuperior olives. This nucleus has been described in cat byOsen et al. (1984) and is equivalent to the dorsal perioli-vary nucleus in the guinea pig as described by Schofieldand Cant (1991). The injected cochlear nucleus itselfalways contained labeled fibers and endings, but wasexcluded from the analysis.

The size of labeled axons was assessed by measuring thediameter of fiber segments proximal to the injected cochlearnucleus before the axons divided into collaterals. There-fore, only intermediate acoustic stria fiber segments lo-cated between the dorsal border of the restiform body andthe ventral border of the spinal trigeminal tract weremeasured. For the trapezoid body, the measurements weremade between the ipsilateral cochlear nucleus and thezone where they began to send off collaterals, ventrolateralto the ipsilateral SOC. For fibers of the lateral tract of thetrapezoid body, measurements were made of the segmentstraveling within that tract, just medial to the cochlearnucleus. For the measurements, a line representing thediameter was drawn perpendicular to the long axis ofeither what appeared to be the thickest part (as forribbon-like segments) or the middle part of the segmentsunder 3100 magnification (oil). Only segments travelingparallel to the plane of section were included and thesewere selected randomly. The resulting drawings werescanned into a computer, and the diameter of the segmentswas then calculated (Designer software; Micrografx, Inc.,Richardson, TX). The means and standard errors of fiberdiameter were calculated and differences between groupstested for significance by the Kolmogorov-Smirnov andKruskal-Wallis ANOVA tests (CSS: Statistica; Statsoft,Tulsa, OK).

RESULTS

The cats were divided into three groups based onlocation of the injection site as defined by the presence ofboth labeled cell bodies and dense background labeling inthe main PVCN regions. In the largest injections, PHA-Llabel was present in both the core and multipolar regionsand thus this group was called the ‘‘core and multipolar’’group. In the ‘‘multipolar’’ and ‘‘core’’ groups, the PHA-Llabel was localized predominately to regions containingmultipolar and octopus cells, respectively. The results ofeach of the three groups, ‘‘core and multipolar,’’ ‘‘multipo-lar,’’ and ‘‘core’’ will be presented separately. The injectionsites will be presented, followed by a description of thetracts containing labeled axons, and then the areas contain-ing labeled varicose fibers. More details are given of the‘‘core and multipolar’’ group because it represents betterthan the other two groups the complete picture of PVCNprojections. Unique cases and special features are given atthe end of the section.

PHA-L injections of both coreand multipolar regions

Figure 1 shows the distribution of PHA-L-labeled cellbodies and dense background staining in the injectedcochlear nucleus coded for each of the six cats of the ‘‘coreand multipolar’’ group. The injection sites were similar inthat each produced labeled neurons in both the core and

multipolar regions. However, other PVCN regions werealso labeled, including the small cell cap and granule celllayer (cats 2433 and 8439) and the anterodorsal region(cats 1044 and 8470). Outside of PVCN, labeled cell bodieswere present in the region of the eighth nerve root (theposteroventral part of AVCN, cat 1044) and in the dorsalcochlear nucleus (DCN; cats 923, 8439, 2433, and 1044). Insections through the injection site of each cat, the morphol-ogy of some labeled cell bodies could be identified asoctopus, multipolar, and various small cell types.

Most of the labeled fibers exited the cochlear nucleus inone of three tracts: the intermediate acoustic stria, thetrapezoid body, or the lateral tract of the trapezoid body.Labeled fibers traveling in the intermediate acoustic striaappeared to be one of three sizes: thin, medium, or thick.Thin-looking fibers had a mean diameter of 2.58 6 0.14µm, medium-sized fibers of 4.65 6 0.11 µm, and thickfibers of 8.09 6 0.19 µm; all fiber types were statisticallysignificantly different in size from each other(H 5 87.02625; df 5 2, n 5 135; P , 0.05).

Based on size, there were two distinct fiber groupstraveling in the trapezoid body. One group consisted ofthin-caliber fibers (2.01 6 0.04 µm) that traveled in ex-treme posterior and ventral parts of the trapezoid body.They passed ventral to the caudal pole of the ipsilateralSOC, entering the ventral nucleus of the trapezoid bodybefore continuing to the other side. Medium-sized trap-ezoid body fibers (3.3 6 0.06 µm) were significantly largerthan the thin ones, and their collaterals appeared toinnervate both the contralateral and ipsilateral SOC. Likethe thin fibers, the medium-sized trapezoid body fiberstraveled in the posterior trapezoid body.

The lateral tract of the trapezoid body, a bundle of fiberssituated just medial to the cochlear nucleus in the ventral-most extreme of the brainstem (described by Warr, 1972),contained labeled axons that were of a thin caliber(2.02 6 0.06 µm). After departing the tract at the level ofthe anterior SOC, the axons (some passing through thelateral pontine nucleus) entered the ipsilateral laterallemniscus. A few axons were observed to directly enter theventral nucleus of the lateral lemniscus. Other axons,after exiting the tract, began traveling medially andcrossed midline in the extreme anterior part of the trap-ezoid body. Upon reaching the opposite side, they enteredthe lateral lemniscus, joining axons of the trapezoid body.

Although a minority, some dorsal acoustic stria fiberswere labeled in the cats in which cells in DCN were alsolabeled. As with most thick fibers, the label in dorsalacoustic stria fibers faded as the distance from the injec-tion site increased, and they could not be traced beyond thelevel of the contralateral ventral nucleus of the laterallemniscus. But before that level, they were not observed tosupply collaterals to the ipsilateral or contralateral SOC.In two cats, labeled axons were observed exiting theventral cochlear nucleus in the vestibular nerve root. Thelabel in those axons, presumably olivocochlear axons,completely faded at the dorsal edge of the spinal trigemi-nal tract.

Besides the traditional routes taken by axons travelingin the intermediate acoustic stria and trapezoid body, a fewindividual axons were observed to take an aberrant course.For example, some axons exited the medial border of thecochlear nucleus, penetrated the inferior cerebellar pe-duncle, and eventually, after traveling medially, joinedfibers from the dorsal part of the trapezoid body. In

PVCN PROJECTIONS 441

another cat, a collateral branched off a medium-sized axonthat was traveling toward midline in the medial nucleus ofthe trapezoid body. The collateral turned to travel backtoward the cochlear nucleus and entered the lateral supe-rior olive after passing through the medial superior olive.

Locations of varicose fibers in cat 2433

A reconstruction (Fig. 2) of the brainstem of cat 2433 isincluded to illustrate all of the projections of the core andmultipolar regions of the PVCN. This cat was selected as arepresentative because the varicose fiber labeling wasfairly robust and the injection site did not enter AVCN. InFigure 2, black-colored areas represent places where la-beled varicose fibers were observed. All parts of theipsilateral SOC contained varicose fibers; the most werelocated in the posterior periolivary nucleus and the lateralnucleus of the trapezoid body. Moderate numbers of vari-cose fibers were located in the mediodorsal (not shown inthe particular sections reconstructed), dorsolateral, andanterolateral periolivary nuclei, as well as the lateralsuperior olive. Varicose fibers were sparse in the dorsome-dial periolivary nucleus, the medial superior olive, and the

ventral and medial nuclei of the trapezoid body (notshown).

In the contralateral SOC, varicose fibers were heaviestin caudal parts of the ventral nucleus of the trapezoid body.An intermediate number of varicosities were located in thecontralateral dorsomedial, mediodorsal, and anterolateralperiolivary nuclei, as well as the lateral nucleus of thetrapezoid body. Although many fewer in number, varicosefibers were observed in the medial nucleus of the trapezoidbody and in the medial superior olive (not shown). Com-pared with the ipsilateral SOC, there were fewer varicosefibers in the contralateral SOC.

Bilaterally, anterior to the SOC, varicose fibers wereobserved in all auditory nuclei, including all three nuclei ofthe lateral lemniscus, the central nucleus of the inferiorcolliculus, the sagulum, and within the lateral part of thelateral lemniscus itself (not shown for the ventral anddorsal nuclei of the lateral lemniscus and sagulum on theright side). Varicose fibers were also observed in theipsilateral lateral pontine nucleus (not shown). In additionto the varicose endings, thick calyceal-type endings wereobserved in the contralateral ventral nucleus of the lateral

Fig. 1. Fifteen sections illustrating the distribution of Phaseolusvulgaris-leucoagglutinin (PHA-L) injection sites in six cats in whichboth the core and multipolar regions of the posteroventral cochlearnucleus (PVCN) were injected. For each cat, the symbol as shown inthe key at the bottom right, represents one labeled cell body. In areas

where individual cell bodies could not be discerned due to densebackground staining, each symbol was adjusted in size to fill the entirearea of background staining. For abbreviations, see list. Scale bar 51 mm.

442 A.M. THOMPSON

lemniscus. For each region above the SOC, the number ofvaricosities and fibers was always greater on the contralat-eral side. Whereas a few labeled axons could be followedwithin the dorsal acoustic stria to locations near thecontralateral cochlear nucleus, varicose fibers were notobserved within the contralateral cochlear nucleus itself.

Locations of varicose fibers of all catsof the ‘‘core and multipolar’’ group

In the ipsilateral SOC and considering the entire ‘‘coreand multipolar’’ group as shown in Table 1 (InjectionC 1 M), varicose fibers were observed in all periolivarynuclei, the ventral and lateral nuclei of the trapezoid body,and the lateral superior olive. Varicose fibers were ob-served in the medial superior olive of five cats and themedial nucleus of the trapezoid body in two cats of thisgroup. The density of varicosities was high in nucleisituated lateral to the medial superior olive and wasespecially heavy in the posterior periolivary nucleus.

In the contralateral SOC (Table 2), varicose fibers wereobserved less frequently. However, they were present inthe ventral and medial nuclei of the trapezoid body, and inthe dorsomedial and anterolateral periolivary nuclei of all

cats. In some cats, varicose fibers were also observed in theremaining periolivary nuclei, as well as in the lateral andmedial superior olives. The varicosities were the mostdense in the ventral nucleus of the trapezoid body. Inaddition to varicose fibers, calyceal endings were alsoobserved in the contralateral medial nucleus of the trap-ezoid body of cat 1044.

In regions of the ipsilateral brainstem above the level ofthe SOC (Table 3), labeled varicose fibers were observed inthe ventral nucleus of the lateral lemniscus of all cats. Thepresence of varicose fibers in other nuclei above the SOCvaried between cats but for the group, varicose fibers werealso observed in the intermediate and dorsal nuclei of thelateral lemniscus, sagulum, and lateral pontine nucleus.

Contralaterally, above the SOC, labeled varicose fiberswere usually observed in all three nuclei of the laterallemniscus, the inferior colliculus, and the sagulum. Vari-cose fibers were not observed in the lateral pontine nucleuson the contralateral side.

PHA-L injections of the multipolar region

Figure 3 shows the injection sites of four cats in whichPHA-L injections were located primarily in the multipolar

Fig. 2. Reconstruction of the brainstem of cat 2433, representingthe group of cats in which both the core and multipolar regions wereinjected with PHA-L. The locations where labeled varicose fibers werefound are indicated by black areas (not intended to represent actual

size of the area). Numbers in parentheses represent the distance inmillimeters from section 1, which is the most posterior. For abbrevia-tions, see list. Scale bar 5 1 mm.

PVCN PROJECTIONS 443

region. Although the morphology of labeled cell bodiescould not always be discerned, multipolar cells of variousshapes and sizes, as well as small cells, could be identified.The injection site was most extensive in cat 3264, andadvanced into the anterior-most border of PVCN. Theinjection site in cat 1155 was the smallest, resulting in thelabeling of only two trapezoid body fibers. Most labeledaxons (both medium and thin caliber) traveled in thetrapezoid body, and, whereas fewer in number, axons werealso observed in the lateral tract of the trapezoid body(2678 and 3264), intermediate acoustic stria (6594 and3264), and dorsal acoustic stria (2678, 1155, and 3264).

In the ipsilateral SOC (Table 1, Injection M), varicosefibers were observed in all of the principal and periolivarynuclei. In the contralateral SOC (Table 2), varicose fiberswere observed in all of the principal and periolivary nuclei,except the lateral superior olive. In the ipsilateral brain-stem, above the SOC (Table 3), they were observed in thethree nuclei of the lateral lemniscus, the inferior collicu-lus, sagulum, and lateral pontine nucleus. Contralaterally,varicose fibers were observed in the same nuclei except forthe lateral pontine nucleus.

PHA-L injections of the core region

Six cats received injections into the core region asdepicted in Figure 4. The core region is known to containmainly octopus cells, but their morphology was not alwaysdiscernible. In some cats, small cells in the region wereclearly labeled. Most of the axons labeled were of mediumand thick caliber and traveled in the intermediate acousticstria. A few labeled axons were also observed in thetrapezoid body of all cats except cat 3568 and in the lateraltract of the trapezoid body of cat 1327.

In the ipsilateral SOC (Table 1, Injection C), varicosefibers were observed in all cats in the posterior andanterolateral periolivary nuclei, and the lateral nucleus ofthe trapezoid body. Whereas they were observed lessfrequently, sparse numbers of varicose fibers were ob-served in the ventral nucleus of the trapezoid body, thedorsomedial and mediodorsal periolivary nuclei, and thelateral superior olive. Furthermore, labeled varicose fiberswere not observed in the medial superior olive or medialnucleus of the trapezoid body. In the contralateral SOC(Table 2), sparse numbers of varicose fibers were observed

TABLE 1. Ipsilateral SOC Regions Containing PHA-L-Labeled Varicosities1

Injection Animal

Periolivary nuclei Principal nuclei

VNTB DMPO MDPO PPO DLPO LNTB ALPO LSO MSO MNTB

C 1 M 923 1 1 1 111 11 1 11 1 1 2C 1 M 5104 1 2 11 111 11 11 11 11 2 2C 1 M 8470 1 1 2 111 11 1 1 1 1 2C 1 M 2433 1 1 11 111 11 111 11 11 1 1C 1 M 8439 1 1 11 111 11 1 11 111 1 1C 1 M 1044 1 1 1 111 11 111 11 111 1 2C 1 M All 1 1 11 111 11 111 11 111 1 1M 6594 1 1 11 111 11 11 11 111 2 2M 1155 1 2 2 1 2 2 1 1 2 2M 2678 1 1 1 111 1 11 11 11 1 2M 3264 11 11 1 111 1 11 11 11 1 1M All 11 11 1 111 11 11 11 111 1 1C 3568 2 1 2 1 2 1 1 2 2 2C 1028 2 2 2 1 1 1 1 1 2 2C 1327 1 1 1 11 11 1 1 2 2 2C 2659 2 2 1 11 2 1 1 2 2 2C 6569 2 1 1 111 11 1 1 1 2 2C 6746 2 2 2 11 1 2 1 1 2 2C All 1 1 1 111 11 1 1 1 2 2All 1 1 11 111 11 111 11 111 1 1

12, 1, 11, and 111 represent increasing numbers of varicosities. For abbreviations, see list.

TABLE 2. Contralateral SOC Regions Containing PHA-L-Labeled Varicosities1

Injection Animal

Periolivary nuclei Principal nuclei

VNTB DMPO MDPO PPO DLPO LNTB ALPO LSO MSO MNTB

C 1 M 923 111 11 2 1 1 2 1 2 2 1C 1 M 5104 111 11 2 2 2 1 11 2 2 1C 1 M 8470 111 11 1 1 2 2 1 2 2 1C 1 M 2433 111 11 11 1 2 11 11 2 1 1C 1 M 8439 111 11 11 1 1 1 1 1 1 11C 1 M 1044 111 111 1 2 1 2 11 2 1 11C 1 M All 111 111 11 1 1 11 11 1 1 11M 6594 111 11 1 1 2 1 1 2 2 11M 1155 11 1 2 2 2 2 2 2 2 2M 2678 11 11 2 1 2 1 11 2 1 11M 3264 111 11 1 1 1 2 11 2 1 1M All 111 11 1 1 1 1 11 2 1 11C 3568 2 1 2 2 2 2 2 2 2 1C 1028 2 2 2 2 2 2 1 2 2 2C 1327 1 1 2 2 2 2 1 2 1 2C 2659 1 2 2 2 2 2 2 2 2 2C 6569 1 2 2 2 2 2 1 2 2 1C 6746 1 1 2 2 2 1 2 2 2 2C All 1 1 2 2 2 1 1 2 1 1All 111 111 11 1 1 11 11 1 1 11

12, 1, 11, and 111 represent increasing numbers of varicosities. For abbreviations, see list.

444 A.M. THOMPSON

in the medial and ventral nuclei of the trapezoid body, thedorsomedial and anterolateral periolivary nucleus, andthe medial superior olive of some of the cats.

Above the level of the SOC ipsilaterally, varicose fiberswere found in the ventral and intermediate nuclei of thelateral lemniscus, as well as in the inferior colliculus.Contralaterally, they were observed in all three nuclei ofthe lateral lemniscus (with calyceal-type endings in theventral nucleus), inferior colliculus, and sagulum. Nofibers were observed in any cats of this group in theipsilateral or contralateral lateral pontine nucleus.

Other observations

In cat 3264 of the ‘‘multipolar’’ group, the PHA-L injec-tion was relatively large, resulting in a relatively largenumber of labeled trapezoid body axons. Relatively morevaricose fibers (both thick and thin) were observed in theposterior parts of both the ipsilateral and contralateralventral nucleus of the trapezoid body and the dorsomedialperiolivary nucleus (Fig. 5). As the fibers streamed be-tween these two periolivary nuclei, they passed within theboundary of the medial nucleus of the trapezoid bodywhere they also contained varicosities. Also, all regionshigher than the SOC, excluding the contralateral lateralpontine nucleus, contained labeled varicose fibers.

Cat 1155 had the smallest injection (Fig. 3) comparedwith other cats in the ‘‘multipolar’’ group and was uniquebecause its brainstem did not contain labeled axons in theintermediate acoustic stria. Originating therefore primar-ily from multipolar cell axons of the trapezoid body,varicose fibers were observed ipsilaterally in the lateralsuperior olive and posterior and anterolateral periolivarynuclei, contralaterally in the dorsomedial periolivarynucleus and ventral nucleus of the lateral lemniscus, andbilaterally in the ventral nucleus of the trapezoid body.

One cat of the ‘‘core’’ group (cat 3568) was unique in thatno labeled axons were observed in its trapezoid body.Therefore, originating primarily from octopus cells whoseaxons traveled in the intermediate acoustic stria, labeledvaricose fibers, although sparse, were observed ipsilater-ally in three periolivary nuclei (dorsomedial, posterior,

anterolateral), the lateral nucleus of the trapezoid body,and the ventral nucleus of the lateral lemniscus. Contralat-erally, varicose fibers were observed in the dorsomedialperiolivary nucleus, the medial nucleus of the trapezoidbody, and the ventral and intermediate nuclei of the laterallemniscus.

Labeled varicose fibers did not appear to be arrangedtopographically within the nuclear and periolivary re-gions, with the exception of the ipsilateral lateral andmedial superior olives. The diagram in Figure 6 maps thelocation of varicose fibers within the ipsilateral lateral andmedial superior olives of one brainstem section of four catsof the ‘‘core and multipolar’’ group. Those cats with PHA-Linjections located more dorsally in PVCN, such as cat1044, tended to have varicose fibers situated more medi-ally in the lateral superior olive and more ventrally in themedial superior olive. In cat 8439, in which the PHA-Linjection was placed in ventral PVCN, labeled varicosefibers were situated in the lateral limb of the lateralsuperior olive and in dorsal parts of the medial superiorolive. As indicated, many varicosities in the ipsilateralmedial superior olive tended to be located on its lateralborder and those in the contralateral medial superiorolive, on its medial border. Additionally, in cats of the ‘‘coreand multipolar’’ group in which fibers in the inferiorcolliculus were well-labeled, parent fibers traveled parallelto each other in the plane of the section (possibly parallelto the isofrequency contours) and gave off short collaterals,associated with clusters of varicosities throughout thecentral nucleus.

Besides a topographic organization to the PVCN projec-tions, there appeared to be an association between PVCNprojections to the primary and periolivary nuclei of theSOC. For example, individual fibers innervated both thelateral superior olive and its adjacent periolivary nuclei.This finding was most evident in cat 1044 in whichindividual fibers could be followed as they traveled be-tween the dorsal part of the medial limb of the lateralsuperior olive and the adjacent part of the dorsolateralperiolivary region as reconstructed in Figure 7.

TABLE 3. Nuclei Anterior to the SOC Containing PHA-L-Labeled Varicosities1

Injection Animal

Ipsilateral Contralateral

VNLL INLL DNLL IC Sag LPN VNLL INLL DNLL IC Sag LPN

C 1 M 9232 1 2 2 2 2 2 1 1 1 2 1 2C 1 M 51042 1 2 2 2 2 2 1 1 1 1 1 2C 1 M 84703 1 1 1 2 2 2 1 1 1 1 1 2C 1 M 24332 1 1 1 1 1 1 1 1 1 1 1 2C 1 M 84392 1 1 1 1 1 1 1 1 1 1 1 2C 1 M 10442 1 2 2 2 2 2 1 1 1 1 1 2C 1 M All 1 1 1 1 1 1 1 1 1 1 1 2M 65942 1 2 1 2 2 2 1 1 1 1 2 2M 11552 2 2 2 2 2 2 1 2 2 2 2 2M 26782 1 1 1 1 2 2 1 1 1 1 2 2M 32643 1 1 1 1 1 1 1 1 1 1 1 2M All 1 1 1 1 1 1 1 1 1 1 1 2C 35683 1 2 2 2 2 2 1 1 2 2 2 2C 10282 1 2 2 2 2 2 1 1 2 2 2 2C 13272 1 1 2 2 2 2 1 1 1 1 1 2C 26593 2 1 2 1 2 2 2 2 1 2 2 2C 65692 1 1 2 2 2 2 1 1 1 1 2 2C 67463 1 2 2 2 2 2 2 1 2 2 2 2C All 1 1 2 1 2 2 1 1 1 1 1 2All 1 1 1 1 1 1 1 1 1 1 1 2

11 and 2 represent presence and absence of varicosities. For abbreviations, see list.2Labeled DAS fibers.3No labeled DAS fibers.

PVCN PROJECTIONS 445

DISCUSSION

PVCN projections to periolivary regions

The current study shows that PVCN neurons project toall periolivary regions situated in the ipsilateral brain-stem. The majority of these projections have been de-scribed previously in cat, including the projections to theposterior, dorsomedial, dorsolateral, and anterolateralperiolivary nuclei, and the ventral and lateral nuclei of thetrapezoid body (Warr, 1969; 1972; 1982; Smith et al.,1993a). The projection to the other periolivary nucleus, themediodorsal periolivary nucleus, located in the dorsalSOC, between the medial limb of the lateral superior oliveand the dorsal pole of the medial superior olive, wasobserved in 69% of the cats.

As for the ipsilateral SOC, all seven periolivary regionsof the contralateral SOC were shown to receive inputfrom the PVCN. This finding is inconsistent with previousreports that have shown PVCN inputs to only threecontralateral periolivary nuclei in cat: the ventralnucleus of the trapezoid body and the dorsomedial and

anterolateral periolivary nuclei (Warr, 1969; 1972; 1982;Smith et al., 1993a). However, a PVCN projection to thecontralateral lateral nucleus of the trapezoid body haspreviously been described in guinea pig (Thompson andThompson, 1987, 1991a). In the current study, PVCNprojections to the remaining three contralateral perioli-vary nuclei (the mediodorsal, posterior, and dorsolateral)were not observed in all cats and when present, wereusually sparse.

The projections of the core and multipolar regions to theipsilateral periolivary nuclei were similar in that bothinnervated primarily posterior and laterally situated perio-livary regions (posterior and dorsolateral periolivary nu-clei and the lateral nucleus of the trapezoid body). Theyboth also innervated, to a lesser extent, the dorsomedialand anterolateral periolivary nuclei. PVCN projections tothe contralateral periolivary nuclei were more evident forthe multipolar region, especially innervating the ventralnucleus of the trapezoid body and the dorsomedial perioli-vary region. Projections from the core region to contralat-

Fig. 3. Distribution of the injection sites in four cats in which thePHA-L injection was located primarily in the multipolar region ofPVCN. As in Figure 1, the symbol as shown in the key at the bottom,represents one labeled cell body, and in areas where individual cell

bodies could not be discerned due to dense background staining, eachsymbol was adjusted in size to fill the entire area. For comparisons, thesections are numbered the same as in Figure 1. For abbreviations, seelist. Scale bar 5 1 mm.

446 A.M. THOMPSON

eral periolivary regions were infrequently observed andvery sparse.

PVCN projections to primary SOC nuclei

The current study in cat supports the finding of PVCNprojections to the ipsilateral lateral and medial superiorolives already described in rodents (Thompson and Thomp-son, 1987; Friauf and Ostwald, 1988). Although furtherstudies will be needed to determine whether the principalcells of these nuclei are postsynaptic targets of the PVCNendings, the patterns observed in the present study arereminiscent of innervation by the AVCN. For example,based on the tonotopic maps provided for the cat cochlearnucleus (Leake and Snyder, 1989) and the cat lateral andmedial superior olives (Tsuchitani and Boudreau, 1966;Guinan et al., 1972), the present study demonstrates thattonotopicity is maintained through the PVCN projections.Also, the circuitous and indirect route by which collateralfibers innervated the ipsilateral lateral superior olive werenot unlike the similar innervation by spherical bushy cellsof the AVCN (Smith et al., 1993b). Finally, the endings inthe ipsilateral and contralateral medial superior oliveswere usually located toward the side of origin, as demon-strated for projections from AVCN (Warr, 1982; Smith etal., 1993b). The projections from PVCN to the primarynuclei may be unlike those from AVCN, however, becausethey appear to arise from the same cells that innervateperiolivary regions. With this in mind, PVCN may notinnervate the principal neurons of the primary nuclei, but

instead, may innervate dendrites of olivocochlear neuronsthat extend into those structures.

Projections from the cochlear nucleus to the contralat-eral medial nucleus of the trapezoid body are known tooriginate from globular cells located in the region of theentering eighth nerve. Globular cell axons travel in thetrapezoid body and terminate as the classic calyx of Heldonto postsynaptic neurons (Harrison and Irving, 1964; vanNoort, 1969; Warr, 1972; 1982; Tolbert et al., 1982; Vaterand Feng, 1990; Kuwabara et al., 1991). In the currentstudy, PVCN globular cells were only labeled in two cats(3264, 1044), and calyceal type endings in the contralat-eral medial nucleus of the trapezoid body were observedonly in cat 1044. Therefore, PVCN must supply anothertype of innervation to the contralateral medial nucleus ofthe trapezoid body as has been previously shown in guineapig (Thompson and Thompson, 1987, 1991a, 1991b). Addi-tionally, Morest (1968) has shown non-calyceal type end-ings in the medial nucleus of the trapezoid body thatoriginate from small axons. The morphology of the fibershe described are similar to some observed in cats of thepresent study in that they were oriented dorsoventrally,had a narrow zone of branching, and extended not onlythrough the medial nucleus of the trapezoid body, butdorsally and ventrally into the dorsomedial and ventrome-dial periolivary regions.

The current study indicates that multipolar, but notoctopus cells, are the source of PVCN projections to theprimary nuclei of the SOC. The lateral superior olive in

Fig. 4. Distribution of the injection sites of six cats in which PHA-Lwas injected primarily in the core, or octopus cell, region of PVCN. Asin Figures 1 and 5, each symbol, as shown in the key at the bottom,represents one labeled cell body. In areas where individual cell bodies

could not be discerned due to dense background staining, each symbolwas adjusted in size to fill the entire area. For comparisons, thesections are numbered the same as in Figure 1. For abbreviations, seelist. Scale bar 5 1 mm.

PVCN PROJECTIONS 447

particular, contained varicose fibers in the ‘‘core’’ casesonly when fibers in the trapezoid body were labeled.Therefore, assuming that axons of most octopus cellstravel in the intermediate acoustic stria, the varicosefibers observed in the lateral superior olive in those casesprobably originated from nonoctopus cells whose axonstraveled in the trapezoid body.

PVCN projections to the nuclei of the laterallemniscus and inferior colliculus

The current study adds further support to the literaturedescriptions of the projection from PVCN to the contralat-eral nuclei of the lateral lemniscus and inferior colliculusin cat (Warr, 1969; 1982; van Noort, 1969; Roth et al., 1978;Adams, 1979; Brunso-Bechtold et al., 1981; Glendenninget al., 1981; Oliver, 1984). Other studies have shown aPVCN projection to the ipsilateral ventral nucleus of thelateral lemniscus and inferior colliculus in cat (Warr, 1969;Adams, 1979; Brunso-Bechtold et al., 1981; Glendenninget al., 1981), and another study supports a PVCN projec-tion to the ipsilateral intermediate nucleus of the laterallemniscus in the mustache bat (Zook and Casseday, 1985).The current study expands these midbrain targets bydemonstrating that the ipsilateral, as well as the contralat-

Fig. 5. Reconstruction of varicose fibers, labeled in one section ofcat 3264, through the posterior ventral and medial nuclei of thetrapezoid body and the dorsomedial periolivary nucleus. The fiberswere supplied by thin and medium-sized axons that originated in themultipolar region of PVCN. The drawing shows that the innervation

pattern was similar on both sides of the brainstem. In more anteriorregions, however, varicose fibers were only found on the contralateralside. The ipsilateral brainstem is depicted to the right of the dottedline and the contralateral brainstem to the left of the dotted line. Forabbreviations, see list. Scale bar 5 40 µm.

Fig. 6. A map showing the distribution of labeled varicose fiberswithin the ipsilateral superior olives to illustrate that PVCN projec-tions are topographic. Each symbol indicates the location of varicosefibers for each of four cats (same code used in Fig. 1). Cats in which theinjections were placed in PVCN more dorsally (such as cat 1044),tended to have varicose fibers situated more medially in the lateralsuperior olive and more ventrally in the medial superior olive. Thedistribution was the opposite for cat 8439, in which the injection waslocated more ventrally in PVCN. Varicose fibers were found in centralregions of the nuclei when injections were placed between the dorsaland ventral extremes (cats 5104 and 8470). For abbreviations, see list.

448 A.M. THOMPSON

eral, dorsal nucleus of the lateral lemniscus receives inputfrom PVCN.

The majority, if not all, of PVCN innervation of theipsilateral nuclei above the level of the intermediatenucleus of the lateral lemniscus arises from the multipolarregion, which may or may not include the small cell cap.The varicose fibers that were observed in the core group ofcats in the same nuclei probably originated from axons ofmultipolar and/or small cells whose axons travel in thetrapezoid body or the lateral tract of the trapezoid body.

PVCN projections to sagulum and lateralpontine nucleus

Another of the new findings of the current study wasthat PVCN projects to the sagulum. One common featurein the cats in which varicosities were found in the contra-lateral sagulum was that all contained labeled axons inthe lateral tract of the trapezoid body. These axons weremost frequently labeled and most numerous when themultipolar region was injected. The sagulum is known toreceive input from the inferior colliculus (Adams, 1979;Brunso-Bechtold et al., 1981; Henkel and Shneiderman,1988) and the auditory cortex (Oliver and Hall, 1978b); itprojects to the ipsilateral medial geniculate body (Oliverand Hall, 1978a; Calford and Aitkin, 1983; Hutson et al.,1991), the inferior colliculus bilaterally, and to the contra-lateral sagulum (Adams, 1979; Moore, 1988; Hutson et al.,1991). Therefore, although previously known to be con-

nected with the auditory forebrain and tectum, the currentstudy shows that the sagulum also receives direct input,bilaterally, although primarily contralaterally, from themultipolar region of PVCN.

It has been previously reported that in rat, PVCNprojects to pontine nuclei; with relatively large PHA-Linjections in the rat PVCN and DCN, Kandler and Herbert(1991) showed projections to pontine nuclei bilaterally. Thecurrent study indicates that the PVCN multipolar regionprovides the input to the ipsilateral lateral pontine nucleusin cat. Furthermore, the small cell cap was also labeled inthe cats in which varicose fibers were found in the ipsilat-eral lateral pontine nucleus, suggesting that small cellsmay also contribute. Like the innervation of the sagulum,the tract containing the axons of these cells may be thelateral tract of the trapezoid body. This was concludedbecause labeled axons were located in this tract in all catsin which varicose fibers were found in the ipsilaterallateral pontine nucleus.

Other known PVCN targets

On the basis of studies by Cant and Gaston (1982) andAdams and Warr (1976), it was anticipated that labeledfibers would be found contralaterally in the anterior partof AVCN, in the core region of PVCN, and/or superficialand fusiform layers of DCN, because those regions receiveinput from the contralateral cochlear nucleus, includingthe PVCN. In the current study, thick fibers were observedto travel in the commissural pathways that connect thetwo cochlear nuclei as described by Cant and Gaston(1982), but, as the label of the fibers faded, they could notbe traced to their terminations. It is also feasible that thecommissural axons labeled in this study were labeled byretrograde transport from the injection site, which couldaccount for the lack of labeled endings in the contralateralcochlear nucleus.

In rat, the lateral paragigantocellularis nucleus has alsobeen shown to receive input from PVCN (Kandler andHerbert, 1991; Bellintani-Guardia et al., 1996). The re-ports of the same projection in cat are conflicting becauseAdams and Warr (1976) and Warr (1972) demonstrated theprojection, whereas Kamiya et al. (1988) found that cells inAVCN, not PVCN, were the source of the cochlear nucleusinputs to the lateral paragigantocellularis nucleus. In thecurrent study, fibers were found in the extreme caudalparts of the SOC, but were not present within the bound-aries of the lateral paragigantocellularis nucleus. Becausemost of PVCN was injected, the current study does notsupport an innervation of the lateral paragigantocellularisnucleus by PVCN in cat.

Comments on tracts and possiblecells of origin

In the intermediate acoustic stria, three sizes of axons(thin, medium, and thick) were observed, suggesting thatat least three different sizes or types of PVCN neuronsfollowed that path. Thick and medium-sized fibers havebeen previously demonstrated in this tract (Warr, 1969),and it is fairly well established that the octopus cell, thepredominate cell type of the core PVCN region, suppliesthick axons to the intermediate acoustic stria (Osen,1969b; Warr, 1969, 1972; van Noort, 1969; Adams andWarr, 1976; Oertel et al., 1990; Golding et al., 1995). Theoctopus cell terminates in all of its targets with varicoseendings except in the ventral nucleus of the lateral lemnis-

Fig. 7. Line drawing of varicose fibers found in the ipsilateralsuperior olivary complex and dorsolateral periolivary nucleus of cat1044. The figure was made by superimposing drawings of fouradjacent sections and illustrates that single fibers innervated both thelateral superior olive and its adjacent periolivary nuclei. For abbrevia-tions, see list. Scale bar 5 20 µm.

PVCN PROJECTIONS 449

cus where its terminations are thick and of the calycealtype (van Noort, 1969; Smith et al., 1993a; Schofield,1995a, 1995b). The calyceal endings observed in thatnucleus in the current study therefore probably belong tooctopus cells. PVCN also contains some large-sized multi-polar cells, and those of the nerve root region have beenshown to send their axons through the intermediateacoustic stria (Smith et al., 1993a), which may have beenanother source of intermediate acoustic stria fibers in thecurrent study.

The other predominate PVCN cell type, the multipolarcell, has been shown to give rise to medium-sized axonsthat travel in the intermediate acoustic stria of the rat(Friauf and Ostwald, 1988). These multipolar cell axons, inaddition to giving rise to varicose type endings, give rise tocalyceal-type endings in the contralateral anterolateralperiolivary nucleus (Friauf and Ostwald, 1988). The contra-lateral anterolateral periolivary nucleus was not observedto contain calyceal type endings in the present study. It ispossible that they were present but were not observed duethe difficulty in achieving dense labeling in calyceal-typestructures. In addition to the octopus and multipolar cells,the small elongate cell as described by Adams and Warr(1976) is also thought to contribute axons to the intermedi-ate acoustic stria and may have been the source of thinfibers observed in that tract in the current study.

As demonstrated here, van Noort (1969) reported that inaddition to the thick fibers arising from globular cells,there were two sizes of fibers, small and medium, thattraveled in the trapezoid body and that originated specifi-cally in PVCN. It is possible that one origin of thesmall-caliber fibers are multipolar cells. Another origin ofthe small-caliber fibers may be the small cells scatteredthroughout PVCN and/or the small cells of the small cellcap. A study of the projections of these particular cell typesin PVCN has not been reported. However, Kim et al. (1995)demonstrated projections from the small cell cap bilater-ally to the ventral and medial nuclei of the trapezoid body,and the ventral and dorsal nuclei of the lateral lemniscus(predominately contralateral), and inferior colliculus (pre-dominately contralateral). Although the source of theterminals in their study was in the small cell cap region ofthe superficial AVCN, because the small cell cap region ishomogenous, we might assume that the projections of thesmall cell cap region of PVCN are similar. Adams (1979)observed small cells in the medial border of PVCN thatmay have been part of the small cell cap and that alsoprojected to the inferior colliculus. These same structures—the ventral and medial nuclei of the trapezoid body, theventral and dorsal nuclei of the lateral, and the inferiorcolliculus—contained labeled varicose fibers in cats inwhich the small cell cap was clearly injected (cats 3264,2433, and 8439).

PVCN multipolar cells have been shown specifically togive rise to axons traveling in the trapezoid body (Smithand Rhode, 1989; Oertel et al., 1990; Smith et al., 1993a;Ostapoff et al., 1994) and are probably the source of themedium-sized fibers observed in that tract in the currentstudy. Octopus cell axons have also been shown to enterthe trapezoid body (Rhode et al., 1983; Ostapoff et al.,1994) and therefore could have also contributed to some ofthose axons in the current study in the cats in which thecore region was injected.

Warr (1972) identified small PVCN fibers traveling inthe lateral tract of the trapezoid body that innervated,

bilaterally, the nuclei of the lateral lemniscus and contra-laterally, the inferior colliculus (Warr, 1982). The currentstudy indicates that the fibers may also innervate, bilater-ally, the sagulum and, ipsilaterally, the inferior colliculus.In one study (Warr, 1972) the fibers were described toinnervate the ipsilateral anterolateral periolivary nucleus,a finding that was not observed in the current study.Therefore, the current study suggests that fibers of thelateral tract of the trapezoid body appear as a separatePVCN pathway that bypasses the SOC and innervatespredominately the contralateral, but also the ipsilateralnuclei of the lateral lemniscus, sagulum, and inferiorcolliculus. They also appear to innervate neurons of theipsilateral pontine nucleus. Although the precise origin ofthe tract remains to be found, based on lesion studies(Warr, 1972), the origin of the fibers may be the ventrome-dial multipolar cell region, including the small cell cap andthe granule cell layer. In the current study, whereaslabeled fibers in the lateral tract of the trapezoid body werefound in most cats, they were more numerous in cats inwhich the PHA-L injection was located more ventrally.

Potential contribution of DCNto the observed projections

All labeled cell bodies of the injected cochlear nucleus,including those of the dorsal cochlear nucleus, were consid-ered part of the injection site. Some of the dorsal cochlearnucleus cell bodies could have been retrogradely labeled bytheir axons passing through the injection site in PVCN. Itwas also evident, however, that some dorsal cochlearnucleus neurons were directly labeled with PHA-L, whichwas expected because of the proximity of the dorsalcochlear nucleus to PVCN and the approach to PVCN thatwas used. Furthermore, axons within the tract by whichdorsal cochlear nucleus neurons exit the cochlear nucleus,the dorsal acoustic stria, were labeled, and therefore, thepotential contribution of dorsal cochlear nucleus neuronsto the endings observed was considered.

As the dorsal acoustic strial axons did not send collater-als to innervate either SOC and as it is fairly wellestablished that DCN does not project to the SOC (seeCant and Casseday, 1986 for a discussion), labeled DCNneurons probably did not contribute to the varicositiesobserved in the SOC bilaterally in the current study.Additionally, the innervation of the contralateral nuclei ofthe lateral lemniscus and the sagulum could be followedfrom parent fibers traveling in the lateral part of thelateral lemniscus where DCN fibers do not travel. Later-ally positioned lateral lemniscus fibers could also be tracedto the contralateral inferior colliculus. Therefore, whereasDCN is known to project to the contralateral nuclei of thelateral lemniscus and inferior colliculus (Osen, 1972; Rothet al., 1978; Adams, 1979; Brunso-Bechtold et al., 1981;Glendenning et al., 1981; Oliver, 1984; Majorossy andKiss, 1994), PVCN could also have contributed to varicosefibers in the contralateral brainstem above the level of theSOC observed in the current study.

Ipsilaterally, no fibers were observed from the dorsalacoustic stria entering the lateral lemniscus; the majorityof varicosities observed could be traced from fibers thattraveled in the lateral tract of the trapezoid body, whichoriginated in PVCN. However, because a projection fromDCN to the ipsilateral inferior colliculus has been de-scribed (Adams, 1979; Brunso-Bechtold et al., 1981), it

450 A.M. THOMPSON

could have been a source of some varicosities observedhere in the present study.

Finally, and most significantly, in one cat (8470), neitherDCN neurons nor dorsal acoustic stria fibers were labeledwith PHA-L. In that cat, varicosities were present bilater-ally in the nuclei of the lateral lemniscus and in thecontralateral inferior colliculus. Therefore, labeling ofDCN neurons may be sufficient but not essential, toobserve projections to these nuclei. It is therefore unlikelythat the labeling of DCN neurons influenced the interpre-tation of the current data.

Technical considerations

As with most tracers, PHA-L can be taken up by fibers ofpassage (Cliffer and Giesler, 1988; Schofield, 1990). In ourexperience, the uptake of PHA-L by fibers of passage isminimized when PHA-L is injected through a small-calibermicropipette tip and when only one penetration is made,as was done in all but 1 of the 16 cats presented. Addition-ally, based on Scofield’s work (1990), we cannot assumethat when PHA-L is taken up by a fiber of passage wellenough to be transported all the way to the terminalendings of that fiber, the PHA-L is also transported therelatively shorter distance, back to the cell body. This leadsto the possibility that PHA-L could have been taken up bycollaterals of AVCN neurons innervating PVCN (bothAVCN multipolar and spherical cells are known to projectto PVCN as shown by Snyder and Leake, 1988) withoutlabeling AVCN cell bodies themselves. Therefore, PHA-Lcould have been taken up by the collaterals, retrogradelytransported to the AVCN axon, and then anterogradelytransported to the terminal endings of those axons inregions outside of cochlear nucleus. For such uptake andtransport to influence the present results, the uptakewould have to have led to anterograde labeling of AVCNaxons and to their terminations. However, labeled fiberstraveling from PVCN to AVCN were not observed. Also,axons departing AVCN or traveling in the anterior part ofthe trapezoid body where AVCN axons are known to travel(Warr, 1966; 1982; van Noort, 1969) were not labeled.

As a retrograde tracer in PVCN, PHA-L could have beentaken up by collaterals of olivocochlear neurons and trans-ported retrogradely to their cell bodies in the SOC (Brownet al., 1988). Neither extra- nor intracellular injections ofhorseradish peroxidase into olivocochlear neurons resultsin labeling of varicose fibers in the SOC. However, whenthe tracer Biocytin, which has characteristics similar toPHA-L, is used to label olivocochlear neurons, varicosefibers, which are collaterals of lateral olivocochlear neu-rons, can be visualized in the lateral superior olive (Brown,1993). It is therefore possible that the varicose fibersobserved in the lateral superior olive in the present studyarise not from PVCN, but from lateral olivocochlear neu-rons. This origin is improbable primarily because PHA-Llabel was not observed in proximal portions of olivo-cochlear axons (the two olivocochlear axons that werelabeled lost their labeling and could not be traced into thebrainstem). Additionally, PHA-L-labeling of the cell bodiesof lateral olivocochlear neurons was not observed. Giventhat neither the proximal axons nor their cell bodies oflateral olivocochlear neurons were labeled, it is unlikelythat the varicosities observed in the current study in thelateral superior olive belonged to lateral olivocochlearneurons.

An attempt was made to keep PHA-L out of AVCN andfiber tracts such as the striae and the eighth nerve root. Byrestricting the injections, the entire part of PVCN was notrepresented. Specifically, the most medial parts of the coreregion and dorsal parts of the multipolar region, includingthe anterodorsal region, were not significantly repre-sented. It is possible that different patterns of terminallabeling could have been observed if these regions weremore heavily injected in the present study. A commonoccurrence was fading of labeled axons, especially thickones, as the distance from their origin in cochlear nucleusincreased. Fading of the label in all axons, but especiallythick ones, is most likely responsible for the reduction inthe observations of labeled fibers, with and without vari-cosities, in some animals in the higher auditory nuclei.

The results of the current and previously publishedstudies show that PVCN projects to a wide variety ofextrinsic targets including the opposite cochlear nucleus,both principal and periolivary nuclei of the SOC, the nucleiof the lateral lemniscus, the inferior colliculus, the sagu-lum, pontine nuclei, and the lateral paragigantocellularisnucleus. Based on the popular concept of cell types havingspecific patterns of projections, that PVCN has so manytargets is not surprising because it contains a variety ofcell types. Regardless of the cell types involved though, thecurrent knowledge of the targets themselves indicates thatPVCN influences all levels of the brainstem ascending anddescending auditory system. PVCN also conveys auditoryinformation to nonauditory regions of the brainstem thatare involved in multisensory processing, the startle reflex,and autonomic function.

ACKNOWLEDGMENTS

Dedicated to the memory of Bruce Masterton. Theauthor thanks Bruce Warr for critical comments on anearly version of the manuscript and Glenn Thompson foreditorial comments.

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