Auditory cortical projections to the cochlear nucleus in guinea pigs

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Hearing Research 199 (2005) 89–102

Auditory cortical projections to the cochlear nucleus in guinea pigs

Brett R. Schofield *, Diana L. Coomes

Department of Anatomical Sciences and Neurobiology, University of Louisville, 500 S. Preston Street, Louisville, KY 40202, USA

Received 29 March 2004; accepted 4 August 2004

Available online 7 October 2004

Abstract

We used anterograde tracing techniques to examine projections from auditory cortex to the cochlear nucleus in guinea pigs. Fol-

lowing injection of dextrans into the temporal cortex, labeled axons were present bilaterally in the cochlear nucleus. The distribution

of boutons within the cochlear nucleus was similar on the two sides. The majority of boutons was usually located on the ipsilateral

side. Most of the boutons were located in the granule cell areas, where many small boutons and a few larger, mossy-type endings

were labeled. Additional small, labeled boutons were found in all layers of the dorsal cochlear nucleus, with the majority located in

the fusiform cell layer. Labeled boutons were also present in the ventral cochlear nucleus, where they were located in the small cell

cap as well as magnocellular parts of both posteroventral and anteroventral cochlear nucleus. Similar results were obtained with

injections restricted to primary auditory cortex or to the dorsocaudal auditory field. The results illustrate direct cortical projections

to the cochlear nucleus that are likely to modulate the activity in a number of ascending auditory pathways.

� 2004 Elsevier B.V. All rights reserved.

Keywords: Descending pathways; Efferent; Olivocochlear; Auditory system

0378-5955/$ - see front matter � 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.heares.2004.08.003

Abbreviations: AC, auditory cortex; A1, primary auditory cortex,

Wallace et al. (2000, 2002); CN, cochlear nucleus; cap, small cell cap;

cnr, cochlear nerve root; d, deep layer of the DCN; DC, dorsocaudal

auditory cortex, Wallace et al. (2000, 2002); DCB, dorsocaudal belt (of

auditory cortex), Wallace et al. (2000, 2002); DCN, dorsal cochlear

nucleus; DRB, dorsorostral belt (of auditory cortex), Wallace et al.

(2000, 2002); f, fusiform cell layer of DCN; g, granule cell area; icp,

inferior cerebellar peduncle; lam, lamina region of granule cell area; m,

molecular layer of the DCN; mcp, middle cerebellar peduncle; oca,

octopus cell area of the VCN; rh, rhinal sulcus; S, small field (of au-

ditory cortex), Wallace et al. (2000, 2002); sca, spherical cell area of

VCN; str, striatum; T, transition zone (of auditory cortex), Wallace

et al. (2000, 2002); tb, trapezoid body; VCB, ventrocaudal belt (of

auditory cortex), Wallace et al. (2000, 2002); VCN, ventral cochlear

nucleus; Vest, vestibular nuclei; vn, vestibular nerve; VRB, ventroros-

tral belt (of auditory cortex), Wallace et al. (2000, 2002); Vtr, spinal

trigeminal tract; wm, subcortical white matter* Corresponding author. Tel.: +1 502 852 5686; fax: +1 502 852

6228.

E-mail address: [email protected] (B.R. Schofield).

1. Introduction

The auditory cortex has long been recognized as the

source of large descending projections to both the thal-

amus and midbrain, where the projections can modify

the responses of subcortical cells to acoustic stimuli (re-viewed by Huffman and Henson, 1990). Recent work in

rats has demonstrated significant cortical projections to

auditory nuclei located below the midbrain, specifically

to the cochlear nucleus, superior olivary complex and

areas associated with the nuclei of the lateral lemniscus

(Feliciano et al., 1995; Weedman and Ryugo, 1996a,b;

Mulders and Robertson, 2000; Doucet et al., 2002). This

work has led to the conclusion that auditory cortex candirectly contact subcollicular auditory circuits, and pre-

sumably modify auditory processing even at these very

early stages of the ascending auditory pathways.

The present study focuses on the cortical projections

to the first station in the central auditory pathways, the

mailto:[email protected]

90 B.R. Schofield, D.L. Coomes / Hearing Research 199 (2005) 89–102

cochlear nuclei (CN). In rats, the cortical projections to

the CN originate from primary auditory cortex and ter-

minate in the granule cell areas, where they contact

granule cell dendrites (Weedman and Ryugo, 1996a).

The granule cells project to fusiform cells of the dorsal

cochlear nucleus (DCN), which project in turn to theinferior colliculus. Thus, cortical projections to the CN

are likely to affect ascending projections from the DCN.

Recent studies suggest that the cortical projection to

the CN may be more extensive in guinea pigs than in rats

(Schofield et al., 2001; Jacomme et al., 2003). Specifically,

the cells of origin are greater in number and have a wider

range of soma sizes in guinea pigs. The projections in rats

may arise only from primary auditory cortex (A1),whereas those in guinea pigs arise from at least two areas

(A1 and the dorsocaudal field). Further, the pathway ter-

minates not only in granule cell areas (similar to rats),

but also in additional areas of the dorsal and ventral

CN (Schofield et al., 2001; Jacomme et al., 2003). These

latter projections could allow cortical axons to influence

CN cell types other than granule cells, and thus to mod-

ulate activity in additional ascending pathways. The pur-pose of the present study was to examine the cortical

projections in guinea pigs and to characterize their distri-

bution and axon morphology in both the ipsilateral and

contralateral CN. We made small injections in some

cases (as done in previous studies) and large injections

in other cases in order to gain an overview of the entire

pathway. The results clarify some differences between

rats and guinea pigs, and suggest that other (apparent)differences are the result of small injection sites.

2. Materials and methods

2.1. Surgery

Experiments were performed on 24 Hartley guineapigs obtained from Charles River Labs (Wilmington,

MA). All procedures were completed in accordance with

the University of Louisville Institutional Animal Care

and Use Committee and the NIH Guidelines. Details

of the surgery were the same as described previously

(e.g., Schofield, 2001). Briefly, the animal was anesthe-

tized with halothane (3.5–4.0% for induction; 2.25–

3.0% for maintenance) in a mixture of oxygen andnitrous oxide. Atropine sulfate was administered (0.08

mg/kg, i.m.) to reduce bronchial secretions, and oph-

thalmic ointment was applied to the eyes to prevent dry-

ing of the cornea. The scalp was shaved and disinfected

with polyhydroxydine solution (Solvay Veterinary, Inc.,

Princeton, NJ). The animal was then placed in a stereo-

taxic frame modified for gas anesthesia. Body tempera-

ture was maintained with a feedback-controlledheating pad. The scalp was incised and the wound mar-

gins were injected with 0.25% bupivacaine, a long-last-

ing local anesthetic (Sensorcaine, Astra Pharmaceutical

Products, Westborough, MA). A dental drill was used

to open the skull over temporal cortex. Following injec-

tion of the tracer (described below), the craniotomy was

covered with Gelfoam (Harvard Bioscience, Inc., Hollis-

ton, MA) and the skin was sutured. The animal was thenreturned to its cage and monitored until it regained the

ability to stand normally. It was then given food and

water and returned to the animal facility.

Three anatomical tracers were used: biotinylated dex-

tran (BDA, 10k MW, Molecular Probes, Eugene, OR),

FluoroRuby (FR, tetramethyl rhodamine dextran, 10k

MW, Molecular Probes, Eugene, OR), and fluorescein

dextran (FD, 10k MW, Molecular Probes, Eugene,OR). All tracers were dissolved in saline (10% weight/

volume). Each tracer was injected with a 10 ll Hamilton

syringe dedicated to use with that tracer. The pseudosyl-

vian sulcus and skull landmarks provided guides for the

location of auditory cortical areas in the guinea pig

(Wallace et al., 2000). Ten animals received injections

into the left cortex, thirteen received injections into the

right cortex, and one received injections of FR andFD on opposite sides. Seven of the animals with injec-

tions into one side received injections of FR and FD

into different locations.

Two different strategies were used to inject tracer over

a wide cortical area. In the first approach, several pene-

trations were made parallel to the surface of temporal

cortex. The insertion point was chosen by eye so that

the needle passed into the deep layers of temporal cortex.The needle was inserted �3.5 mm, where an injection of

0.15 ll was made. Additional injections were made at

multiple points as the needle was withdrawn. The se-

quence was repeated for a total of 3–5 penetrations that

extended over most of temporal cortex. In some cases,

two or more additional sites were injected more dorsally.

For the second approach, the syringe was mounted

orthogonal to the cortical surface. The needle was theninserted 1.8 mm, where 0.15 ll tracer was injected. Mul-

tiple injections (up to 37), spaced �0.5–0.7 mm apart,

were made over a large expanse of temporal cortex.

Tracers were injected into individual cortical areas in

several animals. One animal received 3 closely spaced

injections (0.15 ll each) of FD into the dorsocaudal

auditory field. Two other animals received injections

confined to primary auditory cortex (A1). One of theseanimals received a pair of closely-spaced FR injections

and the other animal received separate injections of

FR and FD, thus yielding three experiments to analyze

A1 projections. For these cases, each injection was 0.15–

0.2 ll in volume.

2.2. Perfusion and processing

Five to 19 days after surgery, the animal was sacri-

ficed by inhalation of carbon dioxide (10 min) or by

B.R. Schofield, D.L. Coomes / Hearing Research 199 (2005) 89–102 91

overdose with sodium pentobarbital (440 mg/kg, i.p.).

Following cessation of breathing and loss of corneal

and withdrawal reflexes, the animal was perfused

through the aorta with Tyrode�s solution (�100 ml) fol-

lowed by fixative. Most cases were processed for light

microscopy, for which the fixative was 300–500 ml 4%paraformaldehyde in phosphate buffer (PB; 0.1 M, pH

7.4) followed by a similar volume of 4% paraformalde-

hyde with 10% sucrose in PB. The brain was removed

and stored overnight at 4 �C in the same fixative with

30% sucrose. One case (GP360) was perfused as de-

scribed above except the fixative was 2% paraformalde-

hyde plus 2% glutaraldehyde. In cases with injections of

fluorescent tracers, the cortical surface was photo-graphed before sectioning to document the location of

the injection site (described below). Brains were cut on

a Vibratome or were frozen and cut on a sliding micro-

tome. In most cases, the cortex was cut in the transverse

plane and the brainstem was cut in the sagittal plane. In

a few cases, the entire brain was sectioned in either the

transverse or horizontal planes. All sections were 50

lm thick. Sections were collected in six series.

2.2.1. Processing of BDA

In cases with BDA injections, alternate sections (3 of

the 6 series) were treated with avidin-biotin-peroxidase

and then stained with diaminobenzidine enhanced with

nickel ammonium sulfate (Adams, 1981). The sections

were then mounted on gelatin-coated slides and air-

dried. One or more series was counterstained with thio-nin for identification of cytoarchitectonic boundaries,

and then all slides were cleared and coverslipped with

DPX (Aldrich Chemical Co., St. Louis, MO).

2.2.2. Processing of fluorescent tracers

In a few cases, all the sections were mounted in serial

order on gelatin-coated slides. More often, 3 evenly-

spaced series were mounted for fluorescence and afourth series was mounted for thionin staining. All sec-

tions were air-dried overnight. Sections for fluorescence

were coverslipped with DPX. Other sections were

stained with thionin, cleared, and coverslipped. In some

cases, the fluorescent tracers were converted to a perma-

nent label by staining overnight with a biotinylated anti-

body (biotinylated goat-antirhodamine or biotinylated

goat-antifluorescein, Vector Laboratories, Burlingame,CA); mixed 1:1000 in 0.01 M phosphate buffer, pH

7.4, 0.9% NaCl plus 1% normal goat serum, then proc-

essed with ABC and diaminobenzidine as described

above for BDA.

2.3. Data analysis and photography

The distribution of labeled boutons was plotted inevery sixth section through the CN with a Zeiss Axio-

plan microscope and Neurolucida reconstruction system

(MicroBrightField, Williston, VT). Subdivisions of the

CN were identified according to Hackney et al. (1990).

Subregions of the granule cell area were identified by

comparison with those described in rats (Mugnaini

et al., 1980). In order to illustrate axonal morphology,

labeled axons were drawn with a camera lucida attachedto the microscope.

In most cases with fluorescent injections, a surface

picture of the cortical injection site was obtained prior

to sectioning the brain. Three separate images were ob-

tained. First, a digital camera (Magnafire, Optronics)

with a 50 mm macro lens was mounted on a copy stand

and used to photograph temporal cortex and a millime-

ter scale under incident light. The brain was then re-pho-tographed through an Axioskop microscope, using a

2.5· objective and a 0.5· adaptor for the Magnafire

camera. One image was obtained with epifluorescence,

and another image was obtained with incident light.

The last image included the millimeter scale, which per-

mitted accurate scaling of the microscopic and macro-

scopic images. The images were aligned with Adobe

Photoshop on a Macintosh G4 computer. We then dig-itized a map of the guinea pig auditory cortical areas

(Wallace et al., 2002) and used Photoshop to overlay

the map onto the photograph of the injection site. The

pseudosylvian and rhinal sulci were used to align the

images. For presentation in black and white, the fluores-

cence image was converted to grayscale and the contrast

was inverted such that the fluorescence appeared black.

Photographs of labeled axons were taken with theAxioskop and Magnafire camera. Adobe Photoshop

was used to adjust contrast and brightness and to add

labels. In some cases, color images were converted to

grayscale and the contrast was inverted to improve vis-

ibility of the labeled structures.

3. Results

All three tracers – FluoroRuby, fluorescein dextran

and biotinylated dextran – gave qualitatively similar re-

sults, though the two fluorescent tracers generally labe-

led more axons. The injections in any given case

typically covered only a portion of temporal cortex

(Fig. 1). The injections typically involved cortical layers

II–V, with layers I and VI included to variable degrees.The injections never extended into the subjacent white

matter (Fig. 1(c)). Across cases, the location varied such

that projections from most or all of the auditory cortical

areas were included (except, perhaps, the most rostro-

dorsal regions). We did not detect any differences related

to the side of injection. In the following section, we de-

scribe the course, distribution and morphology of axons

labeled by large injections into multiple auditory corticalareas. We then briefly describe the results of a few cases

with injections confined to a single area.

Fig. 1. Photomicrographs showing a representative injection site in temporal cortex. (a) Surface view of FluoroRuby injection in case GP346 (see

Section 2 for description of figure production). The contrast of fluorescence image was inverted, making the fluorescence appear black, to enhance

visibility of the injection. Outlines indicate the auditory cortical areas defined by Wallace et al. (2000, 2002). Scale bar = 1 mm. Dorsal is up; rostral is

to the left. (b) Outlines showing identity of auditory cortical areas. See list of abbreviations. (c) Fluorescence photomicrograph of a transverse section

through an injection of FluoroRuby in right temporal cortex, case GP324. Scale bar = 0.5 mm; dorsal is up; lateral is right.


3.1. Course of axons into the CN

On the side ipsilateral to the cortical injection, labeled

axons entered the CN by two routes, similar to those de-

scribed in rats (Feliciano et al., 1995). The majority of

axons entered the CN rostrally at the subpeduncular

part of the granule cell area. The axons coursed caudally

within the granule cell area or traveled more superfi-cially in the DCN. A small number of axons entered

the CN via the ventral acoustic stria/trapezoid body.

On the side contralateral to the cortical injection,

labeled fibers entered the CN by the same routes de-

scribed above, although there was not such a predomi-

nance of fibers in the subpeduncular route.

3.2. Distribution of boutons

The distribution of labeled boutons is illustrated for

one case (Figs. 2 and 3). In this and other cases with

large injections, the majority of boutons were located

on the ipsilateral side. In two cases with the largest

amount of CN labeling overall, we compared the num-

ber of boutons in the plots on the ipsilateral and contra-

lateral sides. The ipsilateral/contralateral ratios in thesecase were 2.3 (case GP360; 22,552 total boutons) and 3.4

(case GP367; 2307 total boutons). While the boutons

differed in number, they were distributed over similar

areas on the two sides in each of the cases. Smaller injec-

tions labeled fewer boutons in the CN but the distribu-

tion was similar to that following larger injections.

However, the relative amount of labeling on the two

sides was much more variable. At one extreme, boutonswere labeled only on the ipsilateral side. In a few cases

with small injection sites, the contralateral side con-

tained the majority of boutons. We never observed a

case in which boutons were present only contralaterally.

There was no apparent correlation between the location

of the injection site and the laterality of the label.

Within the CN, the majority of boutons were located

in the granule cell area. Some of the axons extended to

the caudal tip of the CN, which often contained a dense

collection of labeled boutons (Fig. 2, sections 22 and 25;

Fig. 3, sections 38 and 41). The DCN contained addi-tional boutons in all layers; these were often concen-

trated in the fusiform cell or deep layers. In most

cases, boutons were also present in the ventral cochlear

nucleus (VCN). A common area of concentration was

the small cell cap, which is located in the dorsal margin

of the VCN, subjacent to the granule cell area. In the

remainder of the VCN, boutons were most numerous

in regions that contain multipolar cells and globularbushy cells, less numerous in the spherical cell area

(which contains both spherical bushy cells and multipo-

lar cells), and very rare in the octopus cell area.

3.3. Morphology

Most axons in the granule cell area were thin and had

small en passant and terminal boutons (Fig. 4). A fewaxons were thicker and gave rise to larger endings

(Fig. 4(b)), similar to the mossy endings described for

cortical projections to the CN in rats (Weedman and

Ryugo, 1996a). These endings were observed only in

the granule cell regions. The axons often coursed rostro-

caudally in the sagittal plane (Fig. 5(a), arrowheads).

Equally long stretches of axon were not observed in

transverse sections, suggesting that individual axonsmay innervate a region elongated in the sagittal plane.

As described above, a minority of axons entered the

CN ventrally from the trapezoid body (ventral acoustic

Fig. 2. Plot showing the distribution of labeled cortical boutons in the cochlear nucleus ipsilateral to an injection of FluoroRuby into left temporal

cortex. Each labeled bouton is indicated by a black dot. Parasagittal sections; dorsal is up, rostral is to the right. Sections are numbered from lateral

to medial and are spaced 150 lm apart. Case GP360. Scale bar = 1 mm. See list of abbreviations.


stria) (Fig. 5(a), arrows). These axons could be traced as

far as the small cell cap of the VCN (described below).

The trajectory of the axons at the border of the small cell

cap and the granule cell area suggested that some axons

may enter the granule cell area from this ventral route,

but axon reconstruction would be necessary to confirmthis point.

In the DCN, small endings were present in all layers

(Figs. 6 and 7). In addition, large endings similar to

those in the granule cell area were present in the fusi-

form cell layer (Fig. 7(d), asterisks). Throughout the

DCN, most of the boutons were located in the neuropil

(i.e., not obviously associated with Nissl-stained cell

bodies), although some boutons appeared to be in closecontact with fusiform cell bodies. Some axons could be

traced across the layers (Fig. 7(c) and (d), arrows), pro-

viding boutons to two (or occasionally all three) of the

layers.

In the VCN, most of the boutons were small (Fig. 8)

and were located in the neuropil (i.e., not obviously

associated with Nissl-stained cell bodies). As shown

above (Fig. 5(b)), axons were often present in the small

cell cap. Outside the cap, some axons in the VCN fol-

lowed a trajectory that paralleled the eighth nerve fibers,suggesting that the cortical axons may respect the tono-

topic organization of this area (Fig. 8(a)).

3.4. Injections into individual cortical areas

In order to determine whether individual cortical

areas project to the CN, we made small injections into

primary auditory cortex (A1) or the dorsocaudal field(DC). Fig. 9 shows the results from a case with an injec-

tion restricted to A1. In this case, and others with

presumptive injections in A1, the ventral division of

the medial geniculate nucleus contained labeled cells

Fig. 3. Plot showing the distribution of labeled cortical boutons in the cochlear nucleus contralateral to an injection of FluoroRuby into temporal

cortex. Each labeled bouton is indicated by a black dot. Parasagittal sections; dorsal is up, rostral is to the right. Sections are numbered from lateral

to medial and are spaced 150 lm apart. Case GP360. Scale bar = 1 mm. See list of abbreviations.


restricted primarily to the caudal half of the nucleus.

This pattern of labeling is consistent with an injection

in A1 (Redies et al., 1989b). The number of labeled bou-

tons in the CN was smaller than that following large

injections but the distribution was similar. The two

other experiments with injections confined to A1 pro-

duced similar results.

In one animal, we made an injection restricted to areaDC (Fig. 10). Retrogradely labeled cells in the ventral

medial geniculate were located in the rostral part of

the nucleus, confirming an injection in DC (Redies

et al., 1989b). In this case, labeled boutons were found

ipsilaterally in the granule cell area as well as other parts

of the DCN and VCN. Labeled boutons were not

observed in the contralateral CN in this case.

4. Discussion

The present study is the first to assess the cortical pro-

jections to the CN as revealed by large injections in tem-

poral cortex. The results illustrate that auditory cortex

projects bilaterally to the CN in guinea pigs. The projec-

tions originate from cells in cortical areas A1 and DC.

The majority of axons terminate in the granule cell

areas, with additional terminations in much of the ven-

tral CN and in all layers of the dorsal CN. In the follow-

ing section, we discuss technical issues relevant to our

interpretation of the data. We compare our findings to

previous studies and then consider some of the func-tional implications of this pathway.

The tracers used here can be transported bidirection-

ally, and one must consider the possibility that an axon

is labeled by retrograde transport from the injection site

to a cell body in another region, and then anterogradely

down another branch of the axon. Such collateral labe-

ling would not represent a projection from cells in the

injection site. It is very unlikely that such a mechanismcan account for the labeled axons in the CN in the pre-

sent experiments. Such an event would require that indi-

vidual cells send axon collaterals to both the injection

site – i.e., temporal cortex – and the CN. Our cortical

injections routinely labeled cells in brainstem nuclei

(e.g., raphe nuclei) associated with broad modulation

Fig. 4. Photomicrographs showing labeled cortical axons in the granule cell area of the cochlear nucleus. (a, b) FluoroRuby-labeled axons in

the subpeduncular region of the granule cell area (case GP324). (c) Fluorescein dextran-labeled axons in granule cell lamina (case GP360).

(d) FluoroRuby-labeled axon in granule cell region just dorsal to the anterior ventral cochlear nucleus (line indicates border between the 2 regions).

All sections are parasagittal; dorsal is up, rostral is to the right. Scale bar = 20 lm for (a) and (b); 10 lm for (c) and (d).


of central neural pathways. Some of these nuclei also

project to the CN, but to the best of our knowledge,

individual cells are not known to send collaterals to both

the cortex and the CN.

4.1. Comparisons with previous studies

Jacomme et al. (2003) made small injections of BDA

into the auditory cortex of 5 guinea pigs. They described

projections from A1 and the dorsocaudal field to DCN

layers 2–4 and to the VCN. The present results confirm

their findings and extend them in several ways. First, wefound additional projections to the molecular layer

(layer 1) of the DCN. Second, we describe the distribu-

tion of terminals in the VCN, indicating that the termi-

nals are spread throughout much of the VCN except for

the octopus cell area. Third, we observed a small num-

ber of mossy type endings that were not described in

the previous study. This difference is most likely due

to the sizes of injections and the relative paucity ofmossy endings. The previous study described results

from small injections in 4 animals whereas we observed

the mossy endings only after large injections. Fourth, we

used quantitative methods to address the laterality of

projections in guinea pigs.In both rats and guinea pigs, the granule cell area re-

ceives the majority of cortical terminations (present re-

sults; Feliciano et al., 1995; Weedman and Ryugo,

1996a; Jacomme et al., 2003). The present results con-

firm those of Jacomme et al. (2003) in identifying addi-

tional projections that terminate in the DCN and also in

several parts of the VCN in guinea pigs. Retrograde

tracing studies labeled a morphologically homogeneouspopulation of cortical layer V cells in rats, but a more

heterogeneous population in guinea pigs (Weedman

and Ryugo, 1996b; Schofield et al., 2001). The cells in

rats have large cell bodies and are located deep in layer

V. In guinea pigs, the AC–CN cells range from large to

small, and are scattered throughout layer V. Perhaps the

heterogeneity of cells in guinea pigs is associated with

additional cell types that project to regions outside thegranule cell area.

Fig. 5. (a) Low magnification camera lucida drawing showing the course of labeled cortical axons within the cochlear nucleus. Some axons enter

rostrally and travel caudally through the granule cell area (arrowheads); other axons (arrows) enter the cochlear nucleus ventrally from the trapezoid

body. Box shows location of drawing in (b). (b) High magnification drawings of labeled axons and boutons in the granule cell area and small cell cap.

Parasagittal sections; dorsal is up, rostral is to the right. Scale bar = 1 mm for (a); 50 lm for (b). See list of abbreviations.


A second possibility to explain the differences betweenrats and guinea pigs is a difference in experimental meth-

ods. Feliciano et al. (1995) used PHA-L and biotinylated

dextran, and Weedman and Ryugo (1996a) used biotin-

ylated dextran. The present study also used dextrans

(labeled with biotin or fluorescent markers), suggesting

that the differences are unlikely to be explained by the

tracers. However, other variables, such as transport time

or histological processing, cannot be ruled out as asource of variation. Future experiments will be needed

to discriminate among these possibilities and to identify

the pattern of projections in other species.

4.2. Laterality of projections

The number of labeled boutons in the ipsilateral vs.

the contralateral CN varied from case to case, particu-

larly with the smaller injections. The ipsilateral side of-ten but not always contained the majority of labeled

boutons. A similar result has been described in rats

(Feliciano et al., 1995). Neither study identified a corre-

lation between laterality of projections and the location

of the injection site. In four animals that received re-

stricted injections of BDA into auditory cortex, Jaco-

mme et al. (2003) observed a predominance of

ipsilateral projections in two animals and exclusivelyipsilateral projections in two other animals. The

authors suggested that the laterality of projections

might be related to frequency, with contralateral pro-

jections associated only with low frequency cortical

areas. Our own data (including those obtained with ret-

rograde tracers; Schofield et al., 2001), do not rule out

a frequency bias in the projections, but suggest that

high frequency cortical areas do project to the CN.

Fig. 6. Photomicrographs showing labeled cortical axons in the dorsal cochlear nucleus. (a)–(c) Axons in the molecular layer. Some of the boutons

(arrows) are located close to the surface of the nucleus, which is visible at the upper left of panels (a) and (b). (d) Axons in the fusiform cell layer.

(e) Axons in the deep layer. All panels: Case GP360; FluoroRuby-labeled axons. Parasagittal sections; dorsal is up, rostral is to the right. Scale

bar = 10 lm.


An alternate explanation for differences in laterality of

the projections is that cells that project to one side or

the other are segregated in cortex, such that some injec-

tions include more ipsilaterally-projecting cells while

others include more contralaterally-projecting ones.

Preliminary data, from cases in which we injected dif-

ferent tracers into left and right CN, suggest that ipsi-

laterally-projecting cells are spread relatively evenlyacross temporal cortex whereas the contralaterally-pro-

jecting cells are more clustered (unpublished observa-

tions). Thus, some regions are dominated by

ipsilaterally projecting cells whereas other areas have

a mixture of cells projecting to either side. Large injec-

tions would include ‘‘mixed’’ zones as well as ‘‘ipsilat-

eral only’’ zones, and would label more axons in the

ipsilateral CN. This is consistent with our quantitativefindings that large injections labeled �2–4 times as

many boutons on the ipsilateral side. Small injections

could range from ipsilateral only (as seen in our case

GP371), to a mixed laterality. The potential significance

of mixed versus ipsilateral-only zones may be related to

specific physiological properties of cells that are ar-

ranged in patches in cortex (e.g., Read et al., 2002).

For example, the cortex has segregated zones of cells

that are excited by either ear and cells that are excited

by the contralateral ear and inhibited by the ipsilateral

ear. Perhaps these zones have different patterns of pro-

jections to the CN.

4.3. Origins from different cortical areas

There is now evidence from both anterograde and

retrograde tracing studies in rats and guinea pigs show-

ing projections from A1 to the CN (Feliciano et al.,

1995; Weedman and Ryugo, 1996a,b; Jacomme et al.,

2003; present study). The dorsocaudal auditory field

(DC) also projects to the CN in guinea pigs. AreasA1 and DC have different response properties (Redies

et al., 1989a; Rutkowski et al., 2000; Wallace et al.,

2000, 2002), so we predict that their projections to

the CN serve different functions. Weedman and Ryugo

(1996b) identified area Te1 as the source of projections

Fig. 7. Camera lucida drawings showing morphology of labeled axons and boutons in the dorsal cochlear nucleus. (a, b) Low magnification drawings

showing orientation and locations (boxes) of drawings in (c) and (d), respectively. Scale bar = 1 mm. (c, d) High magnification drawings of labeled

axons. Asterisks (*) indicate particularly large endings. Arrows indicate axons that provide boutons to more than one layer of the dorsal cochlear

nucleus. Scale bar = 50 lm. Case GP360. FluoroRuby-labeled axons. Parasagittal sections; dorsal is up; rostral is to the right. See list of

abbreviations.


to the CN in rats. While Te1 is often considered equal

to A1, physiological studies indicate that Te1 contains

several cortical areas (Doron et al., 2002; Rutkowski

et al., 2003). It is possible that cortical projections to

the CN in rats also include contributions from multiple

areas.

Fig. 8. Photomicrographs of labeled cortical axons in the ventral cochlear nucleus. (a) Labeled axons in the posteroventral cochlear nucleus. The

axon is aligned parallel to the fascicles formed by the auditory nerve fibers (lines). (b) Labeled axons in the anteroventral cochlear nucleus. Scale

bar = 5 lm for (a), 10 lm for (b).


4.4. Functional implications

It will be important in future studies to confirm that

the boutons described here are in fact associated with

synapses and, if so, to identify their postsynaptic targets.

Ultrastructural studies in rats suggest that the AC–CN

terminals are excitatory and that they terminate selec-

tively on granule cell dendrites (Weedman and Ryugo,1996a). While the targets of cortical axons have not been

identified in guinea pigs, the predominance of boutons

in the granule cell areas suggests that granule cells

may be targets in guinea pigs as well as in rats. The gran-

ule cells project directly and indirectly to fusiform cells

in the DCN, which project in turn to the inferior collicu-

lus (reviewed by Oertel and Golding, 1997; Young and

Davis, 2002). The cortical projections are thus in a posi-tion to affect intrinsic CN circuits that could in turn af-

fect the large ascending projection from the DCN to the

IC.

The functions of the smaller cortical projections to

the molecular and deep layers of the DCN are more dif-

ficult to surmise because their cellular targets have not

yet been identified. However, it seems reasonable to pre-

dict that they too will modify activity in the ascendingpathway from the DCN to the inferior colliculus. Inter-

estingly, some axons appeared to provide boutons to

multiple layers in the DCN. Fusiform cells have their

cell body located in the fusiform cell layer and dendrites

that invade the molecular and deep layers. It is conceiv-

able then, that fusiform cells are the targets of many of

the cortical axons. Alternatively, the molecular and deep

layers contain several other cell types, any of whichcould be targeted by the cortical projections.

The present results also demonstrate a cortical pro-

jection to the small cell cap of the VCN. The small cell

cap has numerous properties distinguishing it from the

magnocellular portion of the VCN and associating it

with olivocochlear circuits. Medial olivocochlear cells

send axon branches to outer hair cells in the cochlea

and to the small cell cap (Brown et al., 1988b; Benson

and Brown, 1990). The cap also receives primary affer-

ent input different from that which reaches the magno-

cellular VCN (Brown et al., 1988a; Liberman, 1991;Liberman, 1993). The cap thus appears to integrate

ascending and descending information associated with

the medial olivocochlear system. Furthermore, the cap

is a source of projections to medial olivocochlear cells

(Ye et al., 2000). The present results indicate that the

small cell cap could also integrate cortical inputs, which

may thus contribute to the proposed functions of the

cap and olivocochlear circuits in cochlear gain controland intensity coding (e.g., Kim et al., 1995; Ye et al.,

2000).

Cortical axons also terminate in other parts of the

VCN. These regions do not contain granule cells, indi-

cating that if these cells are targets of cortical projec-

tions in guinea pigs they cannot be the only targets. It

is interesting that some cortical axons in the VCN paral-

lel the course of the auditory nerve fibers, suggestingthat they terminate in a frequency-specific manner.

The distribution of cortical boutons suggests that the

targets are multipolar cells or globular bushy cells.

Globular bushy cells project to a variety of brainstem

targets and appear to be particularly important for bin-

aural processing. Multipolar cells are a heterogeneous

group, and participate in many different projections,

including direct projections to the inferior colliculus,ventral nucleus of the lateral lemniscus, various perioli-

vary nuclei, and the contralateral cochlear nucleus

(Schofield and Cant, 1996a,b, 1997; Doucet and Ryugo,

Fig. 9. Plot showing the distribution of labeled cortical boutons in the cochlear nucleus after an injection of FluoroRuby restricted to area A1 of

right temporal cortex. (a) Line drawing depicting a surface view of the cortical areas as defined by Wallace et al. (2002) and the location of the

injection site (black area). (b, c) Plots showing the distribution of labeled boutons in the cochlear nucleus ipsilateral (b) and contralateral (c) to the

injection. Each labeled bouton is indicated by a black triangle. Transverse sections. Sections in (b) progress from caudal (left) to rostral (right);

sections in (c) progress from rostral to caudal. Distance between sections can be calculated by multiplying the difference between section numbers

(lower left of each section) by 50 lm. Scale bars = 1 mm. See list of abbreviations.


1997, 2003; Alibardi, 1998a,b; Cant and Benson, 2003).

At least some of these projections arise from different

populations of multipolar cells (Cant, 1982; Wenthold,

1987; Schofield and Cant, 1996b; Doucet and Ryugo,

1997, 2003); clearly, understanding the role of cortical

projections will require that the target cells and theirprojections be identified.

4.5. Parallel descending pathways to the CN

The role of direct cortical projections to the CN must

be considered in the context of other descending path-

ways by which higher levels of the auditory system could

affect the CN. Such pathways include descending projec-

tions from the inferior colliculus and the superior oli-

vary complex, each of which receive projections from

the higher centers, including auditory cortex (e.g., Felic-

iano et al., 1995).We propose that the different pathways to the CN

serve different functions. Several types of evidence sup-

port this hypothesis. First, the different pathways have

distinct patterns of termination in the CN, suggesting

that they may affect different CN circuits. As dis-

cussed above, the AC–CN projections frequently con-

(a)

(b)

DCB

VCB

VRB

DRB

SA1

T

DC

Dorsal

Rostral

Dorsal

Rostral

3

15

9

GP371

g

g

vn

m

m

f

f

d

d

VCN

VCN

tb

icp

Vtr

Vtr

mcp

tb

cnr

Fig. 10. Plot showing the distribution of labeled cortical boutons in

the cochlear nucleus after an injection of FluoroRuby into area DC of

left temporal cortex. (a) Line drawing depicting a surface view of the

cortical areas as defined by Wallace et al. (2002) and the location of the

injection site (black area). (b) Plots showing the distribution of labeled

boutons in the cochlear nucleus ipsilateral to the injection. Each

labeled bouton is indicated by a black triangle. Parasagittal sections.

Sections progress from lateral (top) to medial (bottom). Distance

between sections can be calculated by multiplying the difference

between section numbers (lower left of each section) by 50 lm. Scale

bars = 1 mm. See list of abbreviations.


tact granule cells and other cell types in both the DCN

and VCN (at least in guinea pigs). In contrast, projec-

tions from the inferior colliculus terminate only in the

DCN (reviewed by Saldana, 1993). Unlike the corticalprojections, the collicular projections terminate rela-

tively evenly throughout the DCN layers. Finally, the

superior olivary complex terminates throughout the

CN, with relatively heavy projections to all areas (re-

viewed by Spangler and Warr, 1991).

Second, some of the descending projections are prob-

ably excitatory while others are inhibitory. Projections

from the cortex and the inferior colliculus are assumedto be excitatory. Jacomme et al. (2003) described 2 units

in the dorsal CN that were excited by stimulation of ipsi-

lateral auditory cortex, although they could not deter-

mine if the effect was monosynaptic. In contrast,

projections from the superior olivary complex appear

to use a variety of neurotransmitters, including GABA,

glycine, and acetylcholine. These projections probably

exert inhibitory as well as excitatory effects (reviewed

by Helfert and Aschoff, 1997).

Recent studies have implicated descending pathwaysin a wide range of functions, including selective atten-

tion, intensity coding, frequency selectivity, differential

gating of sensory information, sound localization,

adjustment of dynamic range, modulation of temporal

processing, discrimination of speech sounds, and plastic-

ity in functional organization of subcortical structures

(reviewed by Huffman and Henson, 1990; Spangler

and Warr, 1991; Rouiller, 1997; Suga and Ma, 2003).Associating any particular descending pathway with a

particular function will require additional information

on the synaptic organization, neurotransmitters and

physiology of the descending pathways.

Acknowledgement

Special thanks to Mr. Arkadiusz Slusarczyk for ex-

pert technical assistance. This work was supported by

grants NIH DC04391 and DC05277.

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