Separate projections from the inferior colliculus to the cochlear nucleus and thalamus in guinea...

Separate projections from the inferior colliculus to thecochlear nucleus and thalamus in guinea pigs

Diana L. Coomes, Brett R. Schofield *

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

Received 9 October 2003; accepted 8 January 2004

Abstract

We used multiple-labeling techniques with retrograde fluorescent tracers to determine whether individual cells in the inferior

colliculus project to the medial geniculate body (MG) and the cochlear nucleus (CN) in guinea pigs. Four possible projection

patterns were examined: (1) to ipsilateral MG and ipsilateral CN; (2) to ipsilateral MG and contralateral CN; (3) to contralateral

MG and ipsilateral CN; and, (4) to contralateral MG and contralateral CN. Following injections of different tracers into two or

more sites, no inferior collicular cells were double-labeled from the two contralateral targets and only a few cells were double-labeled

from each of the other pairs of targets. The double-labeled cells always totaled <1% of the single-labeled populations. We conclude

that collateral projections from the inferior colliculus to the MG and CN are virtually non-existent. Therefore, the ascending and

descending projections to these targets arise from different cells. These cells could potentially receive different inputs and send

different information to higher or lower centers of the auditory pathway.

� 2004 Elsevier B.V. All rights reserved.

Keywords: Auditory system; Neuroanatomy; Collateral; Efferent; Medial geniculate body

1. Introduction

The inferior colliculus (IC) is a major component of

both the ascending and descending auditory pathways

(Faye-Lund, 1988; Oliver and Huerta, 1992). It is theprimary source of auditory input to the medial genicu-

late body (MG) of the thalamus, and thus carries almost

all auditory information destined to reach cerebral

cortex (Oliver and Huerta, 1992; Winer, 1992). The IC

also sends feedback projections to lower auditory nuclei,

including the cochlear nucleus (CN), the superior oli-

vary complex and the nuclei of the lateral lemniscus

(Huffman and Henson, 1990). These descending pro-jections presumably serve to modify the processing of

incoming auditory information. The IC also contributes

to motor and vocalization functions, in part through

direct projections to the pons, superior colliculus, and

the periaqueductal gray (Huffman and Henson, 1990).

Finally, a large commissural projection connects the left

and right IC and may contribute to many IC functions.

Understanding the functions of these various pathwayswill require, among other things, a determination of

*Corresponding author. Tel.: +1-502-852-5686; fax: +1-502-582-

6228.

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

Abbreviations: AP, anterior pretectal nucleus; bic, brachium of the

inferior colliculus; BIC, nucleus of the brachium of the inferior

colliculus; CN, cochlear nucleus; cp, cerebral peduncle; Cu, cuneate

nucleus; FB, Fast Blue; FD, fluorescein dextran; FG, FluoroGold; FR,

FluoroRuby; GB, green beads; ic, internal capsule; IC, inferior

colliculus; ICc, central nucleus of the inferior colliculus; ICd, dorsal

cortex of the inferior colliculus; icp, inferior cerebellar peduncle; ICx,

external cortex of the inferior colliculus; LD, laterodorsal thalamic

nucleus; LGN, lateral geniculate nucleus; LP, lateral posterior

thalamic nucleus; LRt, lateral reticular nucleus; LSO, lateral superior

olivary nucleus; mcp, middle cerebellar peduncle; MG, medial genic-

ulate body; M5, motor trigeminal nucleus; ot, optic tract; PN,

peripeduncular nucleus; PO, posterior thalamic nuclear group; PPT,

posterior pretectal nucleus; RB, red beads; Rt, reticular thalamic

nucleus; SC, superior colliculus; sca, spherical cell area of the cochlear

nucleus; scp, superior cerebellar peduncle; sp5, spinal trigeminal tract;

Sp5, spinal trigeminal nucleus; SN, substantia nigra; tb, trapezoid

body; VCN, ventral cochlear nucleus; Ve, vestibular nucleus; VL,

ventrolateral thalamic nucleus; VP, ventroposterior thalamic nucleus;

VPM, ventral posteromedial thalamic nucleus; ZI, zona incerta; 5,

trigeminal nucleus; 7, facial nucleus; 7n, facial nerve; 8N, vestibular

nerve

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

doi:10.1016/j.heares.2004.01.009

Hearing Research 191 (2004) 67–78

www.elsevier.com/locate/heares

whether different populations of cells project to each

target or, alternatively, individual cells send axon col-

laterals to multiple targets.

The issue of collateral projections is important with

respect to both inputs and outputs. Collateral projec-tions indicate a common origin from a single population

of cells and thus a common set of inputs. It would be

expected that the same information is then sent to each

target. In contrast, projections that originate from sep-

arate populations of cells could receive different sets of

inputs and send different information to the targets. The

presence of collateral projections suggests a common

function whereas separate projections could supportdifferent functions. Of course, any given set of projec-

tions could include some cells that have collateral pro-

jections and other cells that do not. The presence, and

prominence, of collateral projections must be deter-

mined for a full understanding of any particular circuit.

Only a few studies have examined the possibility of

collateral projections from IC cells. Gonz�alez-Hern�an-dez et al. (1991) used retrograde double-labeling meth-ods in rats and found that up to 30% of cells that project

to the ipsilateral MG also project to the contralateral

IC. The prominence of axonal branching has been fur-

ther demonstrated by intracellular labeling of IC cells

(Oliver et al., 1991; Smith, 1992; Reetz and Ehret, 1999).

In fact, experiments by Oliver et al. (1991) led them to

suggest that collateral projections might be the rule,

rather than the exception.Two recent studies suggest that IC cells have collat-

eral projections to higher and lower centers in the au-

ditory pathways. Reetz and Ehret (1999) stained the

axons of 19 IC cells in mice and found that many had

branches that entered multiple efferent pathways. Of

seven axons that entered the lateral lemniscus (i.e.,

presumptive descending projections), four had branches

that entered the brachium of the IC (i.e., presumptiveascending projections). Malmierca et al. (1996) de-

scribed a similar branching pattern in guinea pigs. A

different conclusion, i.e., that ascending and descending

collaterals are rare, was reached in cats (Hashikawa and

Kawamura, 1983). However, each of these studies is

limited by small sample size. In addition, the intracel-

lular studies (Malmierca et al., 1996; Reetz and Ehret,

1999) were limited by an inability to identify the ex-trinsic targets because the axons were not followed to

their termination.

The purpose of the present study is to address the

issue of ascending and descending collateral projections

from the IC in guinea pigs. Given the possibility of

species differences, as has been described for olivocol-

licular projections (Glendenning and Masterton, 1983;

Willard and Martin, 1984; Schofield and Cant, 1992),there is an advantage to focusing studies on a single

species with the goal of obtaining a complete picture of

the circuitry. We have chosen guinea pigs because they

are a prominent choice for auditory studies and have

been the focus of a large number of previous studies of

collateral projections in the auditory brainstem (e.g.,

Robertson et al., 1987; Aschoff and Ostwald, 1988;

Winter et al., 1989; Schofield, 1991, 1994, 2001, 2002;Schofield and Cant, 1992, 1996, 1999; Schofield and

Perkins, 1999). We used double-labeling techniques,

which allow examination of a large number of cells with

known targets.

2. Materials and methods

Experiments were performed on adult albino guinea

pigs obtained from Charles River Laboratories, Inc.

(Wilmington, MA). All procedures were approved by

the University of Louisville Animal Care and Use

Committee and administered following the National

Institutes of Health guidelines for the care and use of

laboratory animals.

2.1. Surgery and perfusion

Each guinea pig was anesthetized with halothane

(3.5% for induction, 2.5–2.75% for maintenance) in a

mixture of oxygen and nitrous oxide. Prior to surgery,

the animal was premedicated with atropine sulfate (0.08

mg/kg, i.m.). Antibiotic ointment (Neosporin Ophthal-

mic) was placed in each eye to prevent drying of thecornea during anesthesia. The scalp was shaved and

disinfected with Xenodine (polyhydroxydine solution;

Solvay Veterinary, Inc., Princeton, NJ). The animal was

then placed in a stereotaxic holder with the incisor bar

mounted 5.0 mm ventral to the horizontal plane through

interaural zero. Body temperature was maintained with

a feedback-controlled heating pad. An incision was

made in the scalp, and the margins of the incision wereinfiltrated with 0.25% bupivacaine (Sensorcaine; Astra

USA, Inc., Westborough, MA), a long-lasting local

anesthetic. A small hole was then drilled in the skull for

each injection, and tracers were injected into the MG

and CN using stereotaxic coordinates (CN: 2.0–4.0 mm

caudal; 3.6–4.0 mm lateral; 1.4–2.5 mm dorsal, all rel-

ative to interaural zero; MG: 4.3–6.5 rostral; 3.5–4.7

mm lateral; 3.9–4.8 mm dorsal). Each tracer (detailsbelow) was injected with a 10 ll Hamilton syringe that

was used only with that tracer. Injections were made at

1–4 sites within each target. A total volume of 0.3–0.8 llwas injected into each target. Following the injection,

the exposed brain was covered with Gelfoam and the

scalp was sutured. Ketoprofen (3 mg/kg, i.m.) was ad-

ministered immediately after surgery and again a day

later to provide 48 h of post-operative analgesia. Aftersurgery, the animal was placed in a clean cage and

monitored until it regained the ability to stand. The

animal was then returned in its cage to the animal fa-

68 D.L. Coomes, B.R. Schofield / Hearing Research 191 (2004) 67–78

cility. It was checked daily for any signs of abnormal

behavior.

Fast Blue (FB, 5% aqueous solution, Sigma, Inc., St.

Louis, MO), Fluorescein dextran (FD, 10% solution in

saline; molecular weight¼ 10,000: Molecular Probes,Inc., Eugene OR), FluoroRuby (FR, 10% solution in

saline; tetramethylrhodamine dextran, molecular

weight¼ 10,000: Molecular Probes, Inc., Eugene OR),

FluoroGold (FG, 4% aqueous solution; FluoroChrome

Inc., Englewood, CO), or red (RB) or green (GB) latex

microspheres (‘‘beads’’; undiluted; Luma-Fluor, Inc.,

Naples, FL) was injected into the left and right MG and

different tracers were injected into the left and right CNof five guinea pigs to label cells in the IC. Table 1 il-

lustrates what tracers were injected into each location

and the inferior colliculi that were examined for each

case.

After an appropriate interval for transport of the

injected tracers (4–7 days), the animal was sacrificed

with pentobarbital (i.p., 440 mg/kg) or CO2 gas (inha-

lation, 10 min). A thoracotomy was then performed andthe animal was perfused through the aorta with ap-

proximately 100 ml of Tyrode�s solution (pH 7.4), 350

ml of 4% paraformaldehyde in 0.1 M phosphate buffer,

pH 7.4 (PB), and then 350 ml of 4% paraformaldehyde/

10% sucrose in PB. The brain was removed and stored in

4% paraformaldehyde/30% sucrose in PB. The following

day the brain was frozen and cut on a sliding microtome

into 40 or 50 lm sections. The brain from caudal me-dulla to frontal cortex was cut in the transverse or

sagittal plane. Six series of sections were cut. At least 4

series were mounted on gelatin-coated slides and al-

lowed to dry. One series was counterstained with thionin

for identification of cytoarchitectonic borders, and all

sections were then coverslipped with DPX (Aldrich

Chemical Company, Inc., Milwaukee, WI).

2.2. Data analysis

Sections were examined with a fluorescence micro-

scope (Zeiss Axioplan 2). The location and extent of the

injection sites were drawn with a camera lucida, an x-y

plotter attached to the microscope, or a Neurolucida

system (MicroBrightField; Colchester, VT).

To identify cells that contained multiple fluorescent

tracers, every sixth section was viewed at high magnifi-

cation (40� objective). The tracers were visualized

through different filters, facilitating identification of

double-labeled cells. FluoroGold and Fast Blue, whichare both observed with an ultraviolet filter set, were not

used in combination to assess the collateral patterns

under study. The distributions of both single- and

double-labeled cells were plotted with the x-y plotter or

the Neurolucida system. Labeled cells were photo-

graphed with a digital camera (Magnafire, Optronics,

Inc., Goleta, CA). Adobe Photoshop was then used to

adjust brightness, contrast, and color balance. It wasalso used to arrange and label photographs and to

overlay images collected with different fluorescent filters.

Borders between IC subdivisions were drawn with

adjacent thionin sections on a Neurolucida system. The

three main IC regions (the central nucleus, dorsal cortex,

and external cortex) were distinguished according to

criteria described in guinea pigs and rats (Faye-Lund

and Osen, 1985; Malmierca et al., 1995).In every case, labeled cells were counted from every

sixth section throughout the IC. For quantitative anal-

ysis, within each case data for a given tracer were in-

cluded only if the series of sections contained at least 100

cells labeled with that tracer. To determine the per-

centage of double-labeled cells, the number of double-

labeled cells was divided by the sum of cells labeled by a

given tracer. The numbers presented have not been ad-justed for sample size. They have been included to il-

lustrate that numerous cells were labeled for each

projection and demonstrate that the overall percentage

of double-labeled cells is quite small.

3. Results

3.1. Characterization of injection sites

Fig. 1 shows a representative injection into the thal-

amus. The tracer included much of the MG and en-

croached on some of the surrounding nuclei. In a few

cases, the injection included parts of the superior colli-

culus, brachium of the IC, or posterior thalamic nucleus.

Table 1

Summary of injection sites

GP# IC plotted Tracer RCN Tracer LCN Tracer RMG Tracer LMG

246 R FG RB GB –

247 R FG FB GB –

266 R/L FR – FB –

354 R/L – FB FD FR

355 R/L FG FB GB RB

The tracers injected into the right and left cochlear nucleus (CN) and medial geniculate body (MG) are indicated for each case. The right (R) and/

or left (L) inferior colliculus (IC) was analyzed. FB, Fast Blue; FD, fluorescein dextran; FG, FluoroGold; FR, FluoroRuby; GB, green beads, RB,

red beads.

D.L. Coomes, B.R. Schofield / Hearing Research 191 (2004) 67–78 69

Because the IC sends axons to these areas (Andersenet al., 1980), IC cells that project to these regions could

have been labeled. However, the results from these cases

were similar to cases in which injections were confined to

the MG, (i.e., there were extremely few double-labeled

cells), so these cases were included in the analysis.

Following injections into the MG, labeled cells were

present bilaterally in the IC and were more numerous

on the ipsilateral side than the contralateral side. Oneach side, the cells were distributed among all three

subdivisions of the IC (the central nucleus, the dorsal

cortex, and the external cortex). Additional cells were

labeled in other auditory areas, such as temporal cor-

tex, as well as non-auditory structures, such as the su-

perior colliculus, substantia nigra, and zona incerta.

Cells in the latter structures are likely to have been

retrogradely labeled from spread of the tracer into

nuclei surrounding the MG. We will focus only on la-

beled cells within the IC.

Injections into the CN were centered on the dorsalCN and the granule cell areas, which are the primary

targets of projections from the IC (reviewed by Salda~na,1993). Our smallest injection into the CN was confined

to that structure, and included parts of both the dorsal

and ventral subdivisions (Fig. 2(a)). Our largest injection

spread throughout the CN and extended medially and

caudally to invade the inferior cerebellar peduncle and

spinal trigeminal tract and nucleus (Fig. 2(b)). Otherinjections spread into the middle cerebellar peduncle

and/or the vestibular nuclei (not shown). None of the

injections spread into the superior olivary complex or

the lateral lemniscus. IC cells have not been reported to

project to the cerebellum, trigeminal or vestibular nuclei;

thus, the results were unaffected by the spread of the

tracer.

The CN injections labeled cells in auditory structures(e.g., the contralateral cochlear nucleus, the superior

olivary nuclei, and the IC) as well as non-auditory

structures (e.g., the external cuneate and lateral reticular

nucleus). Cells in the latter structures were likely labeled

by tracer taken up by axons in the fiber tracts sur-

rounding the CN. Within the IC, the distribution of

labeled cells matched previous descriptions of this pro-

jection (Schofield, 2001), and will not be described fur-ther.

3.2. Analysis of labeled cells

Fig. 3 illustrates single- and double-labeled cells in the

IC. This figure makes several points. First, the results

are based on numerous tracers and the results were

similar regardless of the tracer used to label a particularpathway. Second, the presence of two different tracers in

a single cell was readily identified (Fig. 3(e), (e�)). Third,single-labeled cells frequently were found in close

proximity to cells that contained a different tracer. This

latter point is important when the projections are or-

ganized topographically, as is the case for the pathways

under study. Intermingling of the labeled cells indicates

that topographically similar parts of each pathway werelabeled.

We examined four possible patterns of collateral

projections from individual IC cells: (1) to the ipsilateral

MG and ipsilateral CN; (2) to the ipsilateral MG and

contralateral CN; (3) to the contralateral MG and ip-

silateral CN; and (4) to the contralateral MG and con-

tralateral CN. For purposes of illustration, we have

chosen one representative case, GP355, in which FastBlue and FluoroGold were injected in the left and right

CN, respectively, and red beads and green beads were

injected into the left and right MG, respectively. We

LGN

MG ic

Rt

PN

BICLP

RtLD

ic

LGN

PN

PN1

2

3

4

5

bic

MG

ic

VP

LP

MG

BIC

LGN LDRt

LP LD Rt

ic

BIC

SCot

MG VPM

SN

SC

PPT

AP

LP LD

ic

Rt

VLVPM

PO

cp SN ZI

VPL

GP354

Lateral

Medialrostral

dorsal

1mm

Fig. 1. Drawings of a representative injection site in the medial ge-

niculate body. The black area indicates a FluoroRuby injection in the

left medial geniculate body of GP354. The distance between sections

can be calculated by multiplying the difference between section num-

bers (at the lower left of each section) by 300 lm. Parasagittal sections.

Scale bar¼ 1 mm. See the list of abbreviations.


describe the results of this case first, and then present a

quantitative summary of the data collected across cases.Figs. 4 and 5 show the distributions of IC cells labeled

with FG, FB and RB in case GP355 (GB-labeled cells

are excluded from the figures for clarity). Fig. 4 shows

the labeled cells in the left IC, and thus illustrates cells

that project to the ipsilateral MG and to either CN.

None of the cells were double-labeled. A few double-

labeled cells were observed in other cases, but the results

suggest that extremely few IC cells project to the ipsi-lateral MG and to either CN.

Fig. 5 illustrates IC cells that project to the contra-

lateral MG as well as to either CN in case GP355 (GB-

labeled cells are excluded for clarity). As was true in the

opposite IC, this IC also contained many single-labeled

cells in each of the major subdivisions. Only one cell was

double-labeled. This cell contained FG and RB, indi-

cating collateral projections to the contralateral MGand ipsilateral CN (star in section 2, Fig. 5). Double-

labeled cells were extremely rare, suggesting that very

few IC cells project to the contralateral MG and to ei-

ther CN.

The case illustrated in Figs. 4 and 5 also contained

cells labeled with green beads injected into the right

MG. These cells were examined for the additional

presence of tracers injected into each CN. In the rightIC, ipsilateral to the GB injection, 3398 IC cells were

labeled with GB; three of these were double-labeled.

Two cells contained GB and FG, indicating projections

to the ipsilateral MG and ipsilateral CN. One cell con-

tained both GB and FB, indicating projections to the

ipsilateral MG and contralateral CN. In the right IC,1147 cells contained GB; none of these were double-

labeled.

Quantitative analysis confirmed that very few IC

cells send collateral projections to the MG and the

CN. We counted the labeled cells in every sixth sec-

tion through six inferior colliculi. A total of 16,525

cells were labeled from MG injections and 12,485 cells

were labeled from CN injections. Of these cells, onlynine were double-labeled. These double-labeled cells

were scattered among the three IC subdivisions. Fig. 6

portrays the percentages of double-labeled cells for the

three projection patterns for which double-labeled cells

were observed. No cells were double-labeled by in-

jections into the contralateral MG and contralateral

CN. For the other three patterns, the double-labeled

cells represented <1% of the single-labeled popula-tions.

4. Discussion

The purpose of the present study was to determine

whether IC projections to theMG and the CN arise from

the same cells or from different cells. Our results indicatethat these projections arise almost exclusively from dif-

ferent populations of cells. These findings complement

those of Hashikawa and Kawamura (1983), who reached

a similar conclusion for projections from the IC to the

sp5

CN

CN

Vesp5

scp

Sp5

icpVe

5

Cu

FB

GP355

FG

rostral

dorsal

1 mm

1

3

5

1

3

sp5

Sp5

7

LSO

7n M5

Ve

LRt

icp

5

mcp

mcp

mcp

mcp

tbVCN

sp5

8N

Left Right

tbicpsp5

CN

(a) (b)

Fig. 2. Drawings of injection sites in the cochlear nucleus. (a) Fast Blue injection site in the left CN. (b) FluoroGold injection site in the right CN.

Injection sites are indicated by the black areas. The distance between sections can be calculated by multiplying the difference between section numbers

(at the lower left of each section) by 300 lm. Parasagittal sections. Case GP355. Scale bar ¼ 1 mm. See the list of abbreviations.


ipsilateralMGand to eitherCN in cats.Wehave extendedtheir findings in several ways. First, we examined the

possibility of collateral projections from IC cells that

project to the contralateral MG. Second, we examined a

larger number of animals and used a wider variety of

tracers, including recently developed ones that, in our

experience, are more sensitive than those that were

available for the earlier study. These issues are particu-

larly important for studies in which the primary finding isone of negative evidence (i.e., the lack of double-labeled

cells), and are discussed in more detail below. Third, we

extended the previous analysis to a second species. Our

results thus broaden the generalization that ascending

and descending projections from the IC arise from dif-ferent cells.

4.1. Technical issues

It is unlikely that any given injection will label every

cell of a specific pathway. To maximize the likelihood of

finding collateral projections, we injected tracers at

multiple sites within each target. In many cases thetracer spread into surrounding structures. This was

particularly true for CN injections; however, the injected

nuclei and tracts around the CN are not known to re-

ceive projections from the IC. Furthermore, the superior

Fig. 3. Digital photomicrographs of labeled cells in the IC. (a)–(d) Fluorescent overlays of single-labeled cells. (a) Cells in the left external cortex of

the inferior colliculus (ICx) of GP354. FluoroRuby (FR)-labeled cells project to the left medial geniculate body (MG), the Fast Blue (FB)-labeled cell

projects to the left CN, and the fluorescein dextran (FD)-labeled cell projects to the right CN. (b) and (c) Cells in the right central nucleus of the

inferior colliculus and ICx, respectively, of GP355. Green bead (GB)-labeled cells project to the right MG, FluoroGold (FG)-labeled cells project to

the right CN, and the FB-labeled cell projects to the left CN. (d) Cells in the right ICx of GP266. FB-labeled cells project to the right MG and FR-

labeled cells project to the right CN. (e) and (e�) Cells in the right ICx of GP266. FB-labeled cells project to the right MG. The FR-labeled cell is

double-labeled with FB (arrowhead) and thus projects to the right CN and the right MG. Sections are parasagittal with dorsal up and rostral right.

Scale bar¼ 10 lm.


ICc

ICd

ICx

ICcICd

ICx

ICx

ICd

ICcICd

ICx

rostral

dorsal

GP355Red Beads (to ipsilateral MG)FluoroGold (to contralateral CN)Fast Blue (to ipsilateral CN)

Left IC

1 3

4 7

Lateral

Medial

Fig. 4. Plots of cells in the left IC that project to the ipsilateral MG (black diamonds), contralateral CN (circles), or ipsilateral CN (gray squares).

Each symbol represents at least one labeled cell. Parasagittal sections. The distance between sections can be calculated by multiplying the difference

between section numbers (at the lower left of each section) by 300 lm. GP355. Scale bar 1¼mm.


ICc

ICd

ICx

ICc

ICd

ICx

ICc

ICd

ICx

Right IC

ICc

ICd

ICx

Red Beads (to contralateral MG)FluoroGold (to ipsilateral CN)Fast Blue (to contralateral CN)FluoroGold/Red beads double label

1

54

2

rostral

dorsal

GP355

Lateral

Medial

Fig. 5. Plots of cells in the right IC that project to the contralateral MG (black diamonds), the ipsilateral CN (circles), or the contralateral CN (gray

squares). The star in section 2 indicates a double-labeled cell that projects to the ipsilateral CN and contralateral MG. Each symbol represents at least

one labeled cell. Parasagittal sections. The distance between sections can be calculated by multiplying the difference between section numbers (at the

lower left of each section) by 300 lm. GP355. Scale bar¼ 1 mm.


olivary complex and lateral lemniscus (which do receiveIC projections) were never included in the CN injection

sites. Consequently, the labeled IC cells almost certainly

project to the CN. Injections into the thalamus spread

into regions surrounding the MG, some of which may

receive IC projections (Andersen et al., 1980). In these

cases, we conclude that the labeled IC cells project to

higher centers (not necessarily the MG). The possibility

that we have labeled more cells than intended bolstersthe significance of negative findings and supports our

conclusion of limited collateral projections.

Of particular concern for identification of collateral

projections is the choice of tracers. Many fluorescent

tracers are available, however some (e.g., propidium

iodide and Evans Blue) are less sensitive than others

(e.g., Aschoff and Holl€ander, 1982). Even the most

sensitive tracers can show different sensitivity when ap-plied to a particular pathway (Richmond et al., 1994;

Choi et al., 2002). We minimized the limitations of in-

dividual tracers by using a wide variety of tracers and by

altering which tracer was used to examine a particular

pathway. It is unlikely that we missed a large portion of

any of the pathways under study. Injections into the CN

labeled cells throughout the IC in a pattern that was

similar across different tracers and that matched previ-ous descriptions of this pathway in guinea pigs (Syka

et al., 1988; Shore et al., 1991; Schofield, 2001). To the

best of our knowledge, the distribution of IC cells that

project to the MG has not been described in guinea pigs.

Our results, based on very large injections, preclude any

detailed description of this projection (e.g., differential

projections from specific IC subdivisions to specific MG

subdivisions), but suggest that the projection is similarto the projections described in numerous other species

(cat: Morest, 1965; Kudo and Niimi, 1978; tree shrew:

Casseday et al., 1976; Oliver and Hall, 1978; rabbit:

Tarlov and Moore, 1966; mustached bat: Wenstrup

et al., 1994). The large injections combined with the

presence of labeled cells throughout the IC suggest that

we did not miss a substantial portion of the ascendingprojection.

Another issue regarding choice of tracers concerns

the ability to recognize multiple tracers within a single

cell. A common combination is Fast Blue and nuclear

yellow. These tracers are visualized simultaneously with

a single filter set and are distinguished by color and by

subcellular distribution: nuclear yellow is generally

confined to the nucleus whereas Fast Blue is containedin the cytoplasm. However, Aschoff and Holl€ander(1982) concluded that these tracers often were more

diffusely localized and could be difficult to distinguish in

double-label experiments. To avoid this difficulty, we

used only combinations of tracers that are visualized

with different filter sets. We have used these same

combinations in the past and identified large numbers of

double-labeled cells in both the auditory and visualsystems (e.g., Hallman et al., 1988; Schofield and Cant,

1999). We conclude that the tracers and their applica-

tion were adequate to identify collateral projections.

Finally, it was important that the same result occur in

multiple animals in which a large number of cells were

labeled. This concern is relevant with respect to the

earlier study by Hashikawa and Kawamura (1983),

whose results were based on a single animal for each ofthe projection patterns examined. For the present study,

we examined over 29,000 cells in six colliculi, providing

strong support for our conclusions. As with any double-

labeling experiment, it is likely that we have underesti-

mated the number of cells with collateral projections.

However, given the arguments discussed above, we

conclude that the number of cells with collateral pro-

jections is quite small, and that the pathways understudy originate largely from different cells.

4.2. Previous studies of IC collateral projections

We return now to a point raised in Section 1 citing

evidence for IC cells with ascending and descending

(a) MG

CN

0.17%(range: 0-0.61%)

0.28%(range: 0-0.93%)

CN

MG

0.02%(range: 0-0.4%)

0.03%(range: 0-0.06%)

(c)MG

CN

0.05%(range: 0-0.18%)

0.15%(range: 0-0.44%)

(b)

Fig. 6. Schematic summarizing the percentages of IC cells that send collaterals to the MG and CN. (a) Percentages of double-labeled cells that project

to the ipsilateral CN and ipsilateral MG. Sample size¼ 5 colliculi. (b) Percentages of double-labeled cells that project to the contralateral CN and

ipsilateral MG. Sample size¼ 5 colliculi. (c) Percentages of double-labeled cells that project to the ipsilateral CN and contralateral MG. Sample

size¼ 3 colliculi. Percentages are shown as an average, with the range indicated beneath. Projections are shown from the left IC for simplicity. The

dotted line indicates midline.


collaterals in rats and guinea pigs (Malmierca et al.,

1996; Reetz and Ehret, 1999). In both studies, the axons

of IC cells were observed to branch within the IC and to

send one branch into the brachium of the IC and one

into the lateral lemniscus. This pattern suggests as-cending and descending collateral projections, but the

targets were not identified because the axons could not

be traced to their destination. These observations were

based on a small sample of cells, but the evidence from

the rats, in particular (Reetz and Ehret, 1999), suggested

that a significant proportion of cells have collateral

projections. If this is the case, the present results suggest

that the branches terminate in some pair of targets otherthan the MG and the CN. Perhaps the ascending branch

terminates in the nucleus of the brachium of the IC,

which was not routinely included in our injection site.

Another possibility is that the descending branch ter-

minates in the superior olivary complex, which receives

a substantial projection from the IC (Huffman and

Henson, 1990; Salda~na, 1993). Further experiments will

be necessary to address these possibilities.Another point raised earlier concerns the ubiquity

of collateral projections from IC cells. Apparently few

cells project to the MG and the CN (present results;

Hashikawa and Kawamura, 1983). Collaterals to the

pons and superior colliculus may also be rare, al-

though this conclusion is based on evidence from a

single animal (Hashikawa and Kawamura, 1983). In

contrast, collateral projections are numerous amongcells that project to the ipsilateral MG and the con-

tralateral IC (Gonz�alez-Hern�andez et al., 1991).

Clearly, collateral projections are more important for

some pathways than for others. Characterization of

these pathways, including their projection patterns as

well as their physiological and neurochemical proper-

ties, will be needed to gain further insight into their

functions.

4.3. Functional implications

As discussed in Section 1, separate cells of origin in-

dicate that two pathways could receive different inputs

and could send different information to their targets.

Many of the inputs to the IC terminate in banded pat-

terns (reviewed by Oliver, 2000). Inputs from differentsources have different degrees of overlap, establishing

‘‘synaptic domains’’ that are characterized by different

combinations of inputs. A cell whose dendrites are re-

stricted to one zone will get different inputs than a cell

whose dendrites are restricted to another zone. To date,

the available data focus on the patterns of termination

of various inputs to the IC with respect to the synaptic

domains. It remains to be determined whether, or how,these domains are related to IC cells that project to a

given extrinsic target. With regard to the current study,

it will be of interest to determine whether cells that

project to the MG occupy the same domains as cells that

project to the CN.

Separate origins of projections also raise the possi-

bility that the pathways send different information to

their targets. This would be an obvious result if the cellsreceive different inputs, but it could also occur if similar

inputs terminate on different cell types that process those

inputs in distinct ways. Multiple cell types have been

distinguished by either morphological or physiological

criteria. Two major classes of IC cells have been iden-

tified morphologically: stellate and disk-shaped (Geniec

and Morest, 1971; Rockel and Jones, 1973a,b; Fitzpa-

trick, 1975; Morest and Oliver, 1984). Both classes havesubtypes, and many of these subtypes appear to project

to the MG (Smith and Spirou, 2002; Oliver, 1984) or the

CN (Schofield, 2001). It remains to be determined

whether the proportions of specific cell types differ for

different pathways. IC cells have also been categorized

physiologically by their responses to acoustic stimuli

(reviewed by Ehret, 1997) or by their intrinsic membrane

properties (Peruzzi et al., 2000; Sivaramakrishnan andOliver, 2001; Bal et al., 2002). Unfortunately, little is

known about how these properties and the associated

physiological classes are related to the extrinsic projec-

tion patterns of IC cells.

Do ascending and descending projections have to

originate from different cells? Given that virtually all

ascending auditory information traverses the IC, the

ascending projections must participate in the majority ofauditory functions. Descending projections are generally

credited with a ‘‘feedback’’ function, whereby higher

levels can influence activity at earlier stages of process-

ing in the ascending auditory pathways. Perhaps the

descending signals are sufficiently different from those in

the ascending pathways that it is necessary to keep the

pathways separate (i.e., no collaterals). It is worth not-

ing that the superior olivary complex, like the IC, is amajor source of ascending and descending projections to

other components of the auditory pathways. A major

target of the ascending olivary projections is the IC, and

these olivocollicular projections arise from different cells

than project to the cochlea or CN (Aschoff and Ostwald,

1988; Schofield, 2002). In other words, ascending and

descending projections from the superior olivary com-

plex arise largely from different populations of cells.While not all ascending and descending pathways have

yet been studied, it appears that separate origins for

ascending and descending projections may be a general

characteristic of auditory pathways.

The present study indicates that ascending and de-

scending projections from the IC originate from differ-

ent cells. As described in Section 1, there are many

additional targets of IC projections, including the su-perior olivary complex, pons and superior colliculus, as

well as a large commissural projection between the two

IC. Clearly, there are many possible combinations of


targets that could potentially receive collateral projec-

tions from individual IC cells. In fact, collateral pro-

jections appear to be prominent in projections to the

MG and the contralateral IC (Gonz�alez-Hern�andezet al., 1991). Identifying the projection patterns and theprominence of collateralization will be an important

step toward characterizing the circuits and identifying

their functions.

Acknowledgements

Special thanks to Ark Slusarczyk for assistance with

histology and manuscript preparation. This study was

supported by NIH DC04391 and DC05277.

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