urocortin-expressing olivocochlear neurons exhibit tonotopic and developmental changes in the...

Urocortin-Expressing Olivocochlear Neurons ExhibitTonotopic and Developmental Changes in theAuditory Brainstem and in the Innervation of theCochlea

Alexander Kaiser,* Olga Alexandrova, and Benedikt Grothe

Division of Neurobiology, Department of Biology II, Ludwig-Maximilians-Universitaet Muenchen, 82152 Martinsried, Germany

ABSTRACTThe mammalian cochlea is under direct control of two

groups of cholinergic auditory brainstem neurons, the

medial and the lateral olivocochlear neurons. The former

modulate the electromechanical amplification in outer hair

cells and the latter the transduction of inner hair cells to

auditory nerve fibers. The lateral olivocochlear neurons

express not only acetylcholine but a variety of co-transmit-

ters including urocortin, which is known to regulate homeo-

static responses related to stress; it may also be related to

the ontogeny of hearing as well as the generation of hear-

ing disorders. In the present study, we investigated the dis-

tribution of urocortin-expressing lateral olivocochlear

neurons and their connectivity and distribution of synaptic

terminals in the cochlea of juvenile and adult gerbils. In

contrast to most other rodents, the gerbil’s audiogram cov-

ers low frequencies similar to humans, although their com-

munication calls are exclusively in the high-frequency

domain. We confirm that in the auditory brainstem urocor-

tin is expressed exclusively in neurons within the lateral

superior olive and their synaptic terminals in the cochlea.

Moreover, we show that in adult gerbils urocortin expres-

sion is restricted to the medial, high-frequency processing,

limb of the lateral superior olive and to the mid and basal

parts of the cochlea. The same pattern is present in juve-

nile gerbils shortly before hearing onset (P 9) but transi-

ently disappears after hearing onset, when urocortin is also

expressed in low-frequency processing regions. These

results suggest a possible role of urocortin in late cochlear

development and in the processing of social calls in adult

animals. J. Comp. Neurol. 519:2758–2778, 2011.

VC 2011 Wiley-Liss, Inc.

INDEXING TERMS: cochlear efferents; lateral olivocochlear neurons; auditory brainstem; urocortin; stress and hearing;

development; hearing onset; inner hair cells; cochlea; tinnitus

The detection, filtering, neuronal processing, and percep-

tion of simple and complex auditory signals in space and

time constitutes a great challenge for the auditory system

and is anatomically reflected in a comparable high number

of brain nuclei along the ascending auditory pathway from

the cochlear nucleus to the auditory cortex. In addition to

these bottom-up pathways, several descending neuronal

connections from different sources enable the brain to mod-

ulate incoming information at a more peripheral level (Suga

et al., 2000; Winer, 2005). One component of this heteroge-

nous descending pathway, the olivocochlear system, con-

sists of two major cell groups within the superior olivary

complex of the brainstem and projects to the sensory epi-

thelium in the cochlea (Warr and Guinan, 1979; Warr et al.,

1986). Together with the myelinated olivocochlear neurons

of the medial system (MOC), which originate from the prox-

imity of the medial superior olive (MSO), within the ventral

nucleus of the trapezoid body (VNTB) region of the ventral

brainstem, the unmyelinated axons of the lateral olivoco-

chlear neurons (LOC), which originate from within or around

the lateral superior olive (LSO) terminate in the synaptic

area of the hair cells of the cochlea (Warr, 1992).

By means of their innervation pattern and origin in the

auditory brainstem, the LOC system comprises two

Additional Supporting Information may be found in the online version ofthis article.

Grant sponsor: Deutsche Forschungsgemeinschaft (DFG); Grant number:GR 1205/13-1.

The first two authors contributed equally to this work.

*CORRESPONDENCE TO: Alexander Kaiser, Division of Neurobiology,Department of Biology II, Ludwig-Maximilians-Universitaet Muenchen,Grosshaderner Strasse 2, 82152 Martinsried, Germany. E-mail: [email protected]

VC 2011 Wiley-Liss, Inc.

Received August 13, 2010; Revised February 10, 2011; Accepted April 1,2011

DOI 10.1002/cne.22650

Published online April 13, 2011 in Wiley Online Library (wileyonlinelibrary.com)

2758 The Journal of Comparative Neurology | Research in Systems Neuroscience 519:2758–2778 (2011)

RESEARCH ARTICLE

separate groups of neurons, one group with a discrete tono-

topic projection to the cochlea and a second group with a

more diffuse projection stemming from the LOC shell neu-

rons (Warr et al., 1997). Whereas the synaptic terminals of

the MOC neurons innervate the outer hair cells directly, the

LOC cells form synapses at the afferent dendrites of type I

spiral ganglion cells underneath the inner hair cells. In con-

trast to the principal neurons of the MSO and LSO and in

accordance with their ontogenetical descent from motor

neurons (Karis et al., 2001; Simmons, 2002), both major

types of olivocochlear neurons use acetylcholine as a syn-

aptic transmitter (Raphael and Altschuler, 2003). However,

only in LOC neurons have a variety of additional cotransmit-

ters, like c-amino butyric acid (GABA), enkephalins, dynor-

phins, calcitonin gene-related peptide (CGRP), and others

been demonstrated (Warr, 1992; Eybalin, 1993; Raphael

and Altschuler, 2003). Upon activation of MOC neurons,

outer hair cell-based cochlear amplification is reduced, as

described in numerous studies (Guinan, 1996).

The physiological significance of the LOC neurons, how-

ever, has been notoriously difficult to demonstrate until

recently. Based on lesioning experiments, Darrow and col-

leagues (2006) suggested that the lateral olivocochlear

feedback loop plays a role in balancing the binaural excit-

ability, which is important for localizing sound in space.

However, in a more recent paper from the same lab (Larsen

and Liberman, 2010), the authors refute the possibility that

LOC neurons have a role in balancing the two ears, thus

leaving the role of those neurons again unclear.

Quite recently, along with the discovery and characteriza-

tion of the stress-related neuropeptide family of urocortins

(Ucn, Ucn 2, and Ucn 3), the expression of Ucn in some

neurons of the LSO, but not in other auditory nuclei, has

been demonstrated (Vaughan et al., 1995; Bittencourt

et al., 1999). Based on the presence of Ucn-immunoreac-

tive (Ucn-IR) cells within the LSO and of Ucn-IR fibers within

the olivocochlear bundle and the inner hair cell region of

the cochlea, it was assumed that these neurons are of an

olivocochlear nature (Vetter et al., 2002). Direct evidence,

including retrograde tracing of olivocochlear neurons from

the cochlea and double labeling with antibodies against

Ucn, was, however, not available. Urocortins are members

of the corticotropin-releasing hormone family of peptides

with a high affinity to corticotropin-releasing factor (CRF)

receptors. They play an important role in balancing behav-

ioral and physiological responses to stress (Reul and Hols-

boer, 2002; De Kloet et al., 2005; Pan and Kastin, 2008).

Behavioral tests that compared wild-type mice with differ-

ent mutants of Ucn-deficient mice revealed an impaired

acoustic startle response and elevated hearing thresholds

for auditory brainstem responses (Vetter et al., 2002; Wang

et al., 2002) in the mutants. It has been suggested that at

least part of these effects might be related to a lack of

release of Ucn in the cochlea during development. In addi-

tion, it is well known that in humans stress can lead to hear-

ing impairments, like hyperacusis, temporary threshold

shift, tinnitus, Meniere’s disease, and vertigo (Mazurek

et al., 2009, 2010), which are routinely treated with gluco-

corticoids (Tahera et al., 2007; Canlon et al., 2007).

In our study we investigate 1) whether the Ucn-IR neu-

rons of the LSO project to the cochlear hair cells (retrograde

tracing experiments); 2) how the Ucn-IR cells are distributed

along the tonotopic gradient within the LSO; 3) how the

Ucn-IR terminals are distributed along the cochlear epithe-

lium; 4) what combinations of transmitter expressions in oli-

vocochlear neurons are present; and 5) whether the distribu-

tion of Ucn-IR in the LSO and cochlea is stable around and

after hearing onset. The last item is particularly important,

as it is generally accepted that the cochlear and auditory

brainstem circuitry is fairly complete around hearing onset

(Simmons, 2002; Kandler et al., 2009). To address these

questions we performed experiments in Mongolian gerbils,

which have, in contrast to most other rodents, a well-docu-

mented low-frequency hearing comparable to that of

humans and other, larger mammalian species.

MATERIALS AND METHODS

Mongolian gerbils (Meriones unguiculates) from our own

breeding colony ranging from postnatal day 9 (P9) to P70

were used for retrograde labeling of olivocochlear efferents

and/or immunohistological characterization of the LSO and

cochlea. All animal procedures were performed in accord-

ance with the German guidelines for the care and use of

laboratory animals as approved by the Regierung of Ober-

bayern (AZ 55.2-1-54-2531-57-05, Bavaria, Germany).

Surgical preparation and tracer injectionA total of six adult gerbils (two females and four males)

ranging from postnatal day 59 to 68 were used for unilateral

injections of small amounts (0.5–1.0 ll) of the retrograde

Abbreviations

BSA bovine serum albuminCGRP calcitonin-gene-related peptideChAT choline acetyltransferaseCRF corticotropin-releasing factorCTB cholera toxin subunit BGABA c-amino butyric acidGAD glutamic acid decarboxylaseIHC inner hair cellIR immunoreactivityISB inner spiral bundleLOC lateral olivocochlear neuronsLSO lateral superior oliveMAP2 microtubule-associated protein 2MOC medial olivocochlear neuronsMSO medial superior oliveOHC outer hair cellP postnatal daySV2 synaptic vesicle of Ommata electric organUcn Urocortin

Changes in urocortinergic olivocochlear neurons

The Journal of Comparative Neurology | Research in Systems Neuroscience 2759

tracers cholera toxin subunit B (CTB) conjugated to either

Alexa 488 (CTB488, five animals, Invitrogen, Carlsbad, CA,

C-34775, 0.5% in saline) or Alexa 594 (CTB A594, one ani-

mal, Invitrogen, C-34777, 1% in saline) into the scala tym-

pani of the cochlea. Prior to the application of tracer solu-

tions, the animals were anesthetized by a standard mixture

of ketamine chloride (20 mg/ml, Ketavet) and xylazine chlo-

ride (0.8 mg/ml, Rompun) in saline with an initial dose of

0.5–0.6 ml/100 g of body mass. Depth of anesthesia was

evaluated by the presence of the paw-pinch reflex, and

additional doses of anesthesia were given when needed.

During surgery the rectal temperature of the experimental

animals was kept constant at 37�C by using a heating blan-

ket and a rectal temperature probe. A small screw was fixed

to the skull above the forebrain by dental glue and con-

nected to a head holder. After the skin and muscles overly-

ing the posterior-lateral skull were retracted, the middle ear

was exposed by an opening lateral of the crus communis.

Subsequently, a small hole was drilled into the scala tym-

pani of the cochlea by slowly rotating a fine scalpel blade by

hand. For local draining of the perilymph, size 40 absorbent

points (for drying the root canal, Roeko, Langenau, Ger-

many) were carefully inserted in this opening without touch-

ing any bone or tissue. Immediately afterward a small

amount of tracer solution was applied manually to the

exposed and drained area of the scala tympani by using an

Eppendorf pipette (2.5 ll maximum volume) and fine tips

that were cut to a proper length. In general, the perilymph

within the cochlea is replaced within a minute; thus the

opening was closed immediately after tracer application by

a customized piece of gelatine and histoacrylic glue (Braun,

Melsungen, Germany). Great care was taken to avoid any

spill of tracer solution outside the opening. Finally, the

retracted muscles were glued to the skull with histoacrylic

glue, the skin was sutured, and the animals were allowed to

recover from anesthesia in their cages.

Immunohistochemistry of brain sectionswith combined tracing

After a survival time of 3–4 days, animals were reanes-

thetized with an overdose of pentobarbital and fixation-

perfused intracardially. Following postfixation and trans-

versal sectioning of the auditory brainstem with a Vibra-

tome (40 lm), one alternate series of sections was

mounted and embedded without further treatment,

whereas two other series were single- or double-stained

for urocortin and/or choline acetyltransferase (ChAT),

respectively (for antibody details, see Table 1).

Immunohistochemistry of brain sectionswithout tracing

A total of 34 gerbils from P9 to P99 were used for immu-

nohistochemistry for different combinations of antibodies

(see below). As described for traced animals, untreated ger-

bils were anesthetized and perfusion-fixed, and brains were

removed and postfixed in 4% paraformaldehyde (PFA) over-

night. After washing in phosphate-buffered saline (PBS; pH

7.4), brains were embedded in agarose and cut into 40-lm-thick sections by using a Vibratome (Leica, Nussloch, Ger-

many). Nonspecific staining of sections was blocked with 1%

bovine serum albumin (BSA) in PBS with 0.5% Triton X-100

for 20 minutes at room temperature. The floating sections

were then incubated for 48 hours at 4�C in the same solu-

tion containing primary antibodies (Table 1). Sections were

washed three times in PBS for 15 minutes and incubated

with secondary antibodies (Molecular Probes/Invitrogen,

Karlsruhe, Germany, A21202 1:200, A-21206 1:200,

A11055 1:400, A11015 1:400, A11039 1:400; Dianova,

Hamburg, Germany, 715-166-151 1:400, 715-176-150

1:200, 715-156-150 1:100, 711-165-152 1:400, 705-166-

147 1:400, 703-176-155, 1:200, 703-156-155 1:100;

Abcam Cambridge, UK, Ab6566 1:100) in blocking solution

TABLE 1.

Sources and Dilutions of Antibodies Used in the Study

Antibody Immunogen Manufacturer Host Cat. no. Dilution

Calretinin Recombinant human calretinin-22k SWANT, Bellinzona, Switzerland Mouse monoclonal 6B3 1:500ChAT Human placental enzyme Millipore, Bedford, MA Goat polyclonal AB144P 1:500CGRP Synthetic peptide (rat) conjugated to

BSA by a glutaraldehyde linker.Novus Biologicals Littleton, CO Sheep polyclonal NB600-1156 1:1,000

GABA GABA conjugated to BSA SWANT Mouse monoclonal mAB 3D5 1:300GAD65 Purified rat brain glutamic acid decarboxylase Millipore Mouse monoclonal MAB351R 1:500GAD67 Synthetic peptide: RFRRTETDFSNLFARDLLPA,

corresponding to amino acids 87–106of human GAD67

Abcam, Cambridge, UK Mouse monoclonal ab26116 1:200

MAP2 Bovine MAP2 Neuromics, Edina, MN Chicken polyclonal CH22103 1:1,000SV2 Synaptic vesicles of Ommata electric organ Developmental Hybridoma

Bank of the University of IowaMouse monoclonal SV2-a1 1:500

UCN Peptide corresponding to C-terminal urocortinamino acid sequence 25–40

Sigma, St. Louis, MO Rabbit polyclonal U4757 1:2,000

For abbreviations, see list.

Kaiser et al.

2760 The Journal of Comparative Neurology |Research in Systems Neuroscience

for 3 hours at 37�C. After washing in PBS (3X 15 minutes),

sections were counterstained (when necessary) with a Neu-

roTrace fluorescent Nissl stain (Invitrogen). Finally, sections

were mounted in Vectashield antifade mounting medium

(Vector, Burlingame, CA) and sealed with nail polish.

Immunohistochemistry of cochleaeCochleae for further immunohistochemistry (n ¼ 34)

were taken from the same gerbils that were used for

immunostaining of the LSO.

Immediately after perfusion, both cochleae were

removed from the skull, and the stapes was taken out for

better access of fixative. After 1–2 days of postfixation in

4% PFA, the cochleae were washed in PBS and the tectorial

membrane was exposed by carefully removing abneural

bone and tissue of the perilymphatic tubes in wholemount

preparations. Finally, the basilar membrane was removed

with fine forceps, and the specimens were decalcified for

2–3 days in EDTA solution (7.5% EDTA in 0.8 N NaOH solu-

tion, pH 7.4). Each cochlea was then treated for immuno-

histochemistry separately with different sets of primary and

secondary antibodies (Table 1). Staining procedures were

the same as for the brain sections; however, incubation

time in the primary antibodies was prolonged up to 72

hours at 4�C and 24–48 hours for secondary antibodies.

Following immunostaining, cochleae were serially cut into

three to five pieces, mounted serially on slides using a

stereo-dissecting microscope, and coverslipped with Aqua-

Poly/Mount (Polysciences, Warrington, PA) for further anal-

ysis with epi- and confocal fluorescent microscopy.

Characterization of antibodiesAll primary antibodies are listed in Table 1.

For calretinin, a monoclonal antibody from SWANT

(Bellinzona, Switzerland, 6B3) was applied. Our Western

blot analysis of gerbil brain lysate showed that antibodies

directed against calretinin recognized the single expected

band at�32 kDa.

For ChAT, a polyclonal antibody (AB144P; Millipore,

Bedford, MA,) was used that recognizes the single

expected band at �70 kDa.

Polyclonal antibodies applied for identification of CGRP

(NB600-1156, Novus Biologicals Littleton, CO) recog-

nized the expected band at�3 kDa.

The mouse monoclonal antibody to GABA (mAB 3D5,

SWANT) was evaluated for activity and specificity by blot

immunoassays by the supplier (Matute and Streit, 1986).

No (or very weak) cross-reaction with BSA conjugates was

observed with enzyme-linked immunosorbent assay (ELISA)

for alanine, glycine, taurine, glutamate, and glutamine.

Antibodies directed against GAD65 (MAB351R, Milli-

pore) recognized the expected band at �65 kDa and an

additional lighter band at 72 kDa.

Antibodies directed against GAD67 (ab26116, Abcam)

recognized a single expected band at�70 kDa,

Antibodies directed against MAP2 (microtubule-associ-

ated protein 2) are shown to detect MAP-2a, -b (�280

kDa), and c (�70 kDa) variants in Western blot analysis

(data sheet and personal communication from Neuro-

mics, Edina, MN). This was confirmed in immunocyto-

chemical control experiments carried out by our group on

Mongolian gerbil brain and previously published in this

journal (Rautenberg et al., 2009).

Antibodies directed against the transmembrane glyco-

protein SV2 (Developmental Hybridoma Bank of the Uni-

versity of Iowa) recognized the single significant expected

band at �100 kDa. As expected because of the known

challenges of resolving glycosylated proteins electrophor-

etically (Bajjalieh et al., 1992; Feany et al., 1992; Son

et al., 2000; Nealen, 2005), light bands were distributed

over a range of protein sizes (72–200 kDa).

For the identification of Ucn-containing neurons, we

applied a polyclonal antibody (U-4757; Sigma, St. Louis,

MO). According to the manufacturer’s technical information,

immunostaining for Ucn is specifically inhibited by Ucn, and

the antibody does not cross-react with human or rat CRF

(6-33), sheep CRF, PACAP38, human adrenocorticotropic

hormone (ACTH), human ACTH (18–39), and sauvagine. As

summarized by Vasconcelos et al. (2003), amino acids 25–

40 of the synthetic urocortin is a highly conserved sequence

that is identical in rat, human, sheep, and mouse. In addi-

tion, amino acids 25–36 are also identical for monkey and

sheep. In the rat and the cebus monkey, Ucn staining of

cells was confirmed with Ucn mRNA expression (Bittencourt

et al., 1999; Vasconcelos et al., 2003). In our study the pat-

tern of immunostaining in neurons of the Edinger-Westphal

nucleus, which is generally accepted as a reference nucleus

for Ucn, was identical to recently published staining data in

rat, mouse, ferret, monkey, and human (Vasconcelos et al.,

2003; Horn et al., 2008, 2009; Gaszner et al., 2009).

Furthermore, we observed Ucn-IR in a variety of char-

acteristic cells within the nucleus facialis, the nucleus

hypoglossus, the Raphe nucleus, and cells of lung tissue.

However, the most intense Ucn staining was always

observed in neurons of the LSO and the Edinger-Westphal

nucleus. Intense staining of fibers with Ucn has been

observed in many brain regions, as described in the refer-

ences cited above, e.g., intense Ucn fiber networks were

found in the pituitary gland and parts of the cerebellum.

We routinely carry out control experiments in which we

test our secondary antibodies by omitting the primary anti-

body. Application of secondary antibodies only showed no

staining pattern (data not shown). To confirm the specificity

of double- and triple-immunolabeling experiments, we

compared double/triple-stained brain sections with single

stained ones. We showed that application of several primary



antibodies simultaneously did not change the resulting pat-

tern of single antigen distribution (data not shown). We also

tested the specificity of immunolabeling by omitting the pri-

mary antibodies raised against the second/third antigen in

our scheme and applying only corresponding secondary anti-

bodies. In this case second/third secondary antibodies dem-

onstrated no staining pattern.

Confocal microscopyConfocal optical sections were acquired with a Leica TCS

SP confocal laser-scanning microscope (Leica Microsys-

tems, Mannheim, Germany) equipped with Plan Fl25�/

0.75 NA, Plan 63�/NA1.32, and Plan Apo 100�/1.4 NA

oil immersion objectives. Fluorochromes were visualized by

using an argon laser with excitation wavelengths of 488 nm,

emission 510–540 nm for Alexa 488, a DPSS laser with a

laser line of 561 nm (emission 565–600) for Cy3, and a he-

lium-neon laser with an excitation wavelength of 633 nm

(emission 640–760 nm) for Nissl Deep Red and Cy5. For

each optical section the images were collected sequentially

for two or three fluorochromes. Stacks of eight-bit grayscale

images were obtained with axial distances of 0.3–3 lmbetween optical sections and pixel sizes of 781–100 nm

depending on the selected zoom factor and objective. To

obtain an improved signal-to-noise ratio each section image

was averaged from four successive scans. After stack ac-

quisition, Z chromatic shift between color channels was cor-

rected. RGB stacks, montages of RGB optical sections, and

maximum-intensity projections were assembled into tables

by using ImageJ 1.37k plugins and Adobe Photoshop 8.0.1

(Adobe Systems, San Jose, CA) software.

Quantification of IR cells within the LSOFor quantification of developmental changes in the

LSO and cochlea, four groups of different ages were ana-

lyzed: pre-hearing (P9, n ¼ 4), immediately post-hearing

(P14, n ¼ 4), young post-hearing (P21–P26, n ¼ 4), and

adult (P40–P99, n¼ 5).

In order to estimate the number of cells of the LSO

that were immunoreactive for Ucn, ChAT, and CGRP as

well as the number of cells expressing Ucn/ChAT or

Ucn/CGRP simultaneously, the sections were additionally

counterstained with fluorescent Nissl stain or alterna-

tively with antibodies directed against MAP2, specifically

labeling somata and dendrites of neurons in order to

define the outline of superior olivary structures.

Ucn, ChAT, and CGRP are distributed in the cytoplasm

of cells but not in the cell nucleus (see Fig. 2E,G). The nu-

cleus is therefore visible in the cell somata region as a

negatively stained area. Using the ImageJ plugin Cell

Counter, we manually selected only ‘‘nucleus-containing’’

cells for counting, independently from the cell/nucleus

size, which is variable even within the LSO of gerbils of

the same age, with the maximum diameter of the cell

somata ranging from 10 to 25 lm.

Neuronal tissue tends to shrink when fixed with PFA,

and this shrinkage can be age dependent due to different

myelinization rates in the brain. The size of the LSO

nucleus increases during development, so cell density

decreases with age. Therefore, for analyzing changes in

the amount of Ucn-IR LOC cells throughout developmen-

tal stages, we did not consider absolute cell numbers per

square millimeter, but rather the proportion of Ucn-IR

cells relative to the total ChAT-IR cell population. As all

Ucn-IR cells are also positively immunolabeled with anti-

bodies directed against ChAT (see Results), this normal-

ization proved to be the superior method for comparing

cell counts during development.

Analysis of sections along the rostrocaudal extent of the

LSO nucleus of single gerbils has shown that the relative

amount of ChAT-IR cells (considered as number of cells for

the unit of area or as percentage of cells with other immu-

noreactivity) does not change significantly from section to

section (data not shown). Therefore, for comparing cell

counts in gerbils of different ages, we used only four repre-

sentative Vibratome sections for data evaluation.

Within the posterior-anterior Vibratome section series,

the first section in which the LSO was represented with

both lateral and medial parts was defined as section 1. In

order to adjust for the increase in LSO volume during de-

velopment (Sanes et al., 1989), for each age group we ana-

lyzed four sections with increasing distance from posterior

to anterior with section 1 as a reference. Thus, section dis-

tances were 0, 80, 160, and 240 lm for P9, 0, 120, 240,

and 360 lm for P14, and 0, 80, 160, and 440 lm for

young to adult animal age groups (P21–P63). Stacks of

confocal images were obtained with a step size of 3 lm.The distance between single optical sections was therefore

smaller than the cell size, which prevented loss of informa-

tion. Maximum projection images of confocal stacks

acquired through the LSO were stitched in Adobe Photo-

shop into a single maximum projection image representing

the whole LSO area of a given Vibratome section.

After the borders of the LSO were defined, maximum

projection images of four Vibratome sections from each

gerbil were aligned along the anterior-posterior axis of

the brainstem.

The total area of the LSO was subdivided into 10 arbi-

trary segments along its tonotopic axis from lateral to

medal limb, and the segments were named L5, L4, L3,

L2, L1, M1, M2, M3, M4, and M5 (Fig. 1D). Measurements

of segment area and Ucn, ChAT, and CGRP cell counts

were carried out by using ImageJ plugins. The morphologi-

cal uniformity among LSO nuclei from animals of the

same age (Sanes et al., 1989) suggested that we could

average the data obtained from defined LSO regions for

Kaiser et al.


the gerbils of a given age group. Error bars in all diagrams

used for illustration represent standard errors of the

mean. For cell counts we choose only neurons containing

the visible cell nucleus, which was defined as an area free

of ChAT/Ucn within the cytoplasm. In order to adjust our

profile counts for overcounting errors, cell numbers were

Figure 1. Immunohistochemical characterization of coronal Vibratome sections from adult mongolian gerbils with antibodies against ChAT and uro-

cortin reveals a topographic gradient within the LSO. A: Control for urocortin immunoreactivity. Anti-urocortin antibodies positively stain neurons of

non-preganglionic Edinger-Westphal nucleus. Maximum projection of several optical sections. B: Control for ChAT immunoreactivity. Large cholinergic

motoneurons of the facial nucleus show bright immunofluorescence after applying anti-ChAT antibodies. Maximum projection of several optical sec-

tions. C: Overlay of maximum projections of several optical sections obtained from the LSO double-labeled with anti-urocortin (red) and anti-ChAT

(green) antibodies. Dashed line shows the borders of the LSO as depicted by anti-MAP2 immunostaining (not shown). D: Schematic drawing of the

same section of the LSO as in C including the segments along the tonotopic axis, which were introduced for counting the number of Ucn- and

ChAT-IR cells. L5–L1 comprise the lateral and M1–M5 the medial limbs of the LSO. Orientation of sections as indicated in the lower left corner is

valid for all images. E,F: Corresponding images of Ucn (E) and ChAT (F) immunostaining of the LSO shown in C. Note that Ucn-IR is restricted to

the medial limb of the LSO, whereas ChAT IR cells are present in both medial and lateral limbs of the LSO. All Ucn-IR cells also contain ChAT. G:

Quantification of changes in the number of Ucn- and ChAT-IR cells per square millimeter along the tonotopic axis of the LSO for adult (P40–P63)

gerbils (n ¼ 4) using the segmentation shown in D. ChAT-IR neurons were present throughout the segments, and Ucn-IR cells were almost entirely

restricted to the medial segments (M1–M5). H: Percentage of cells double-labeled for Ucn and ChAT relative to the total number of ChAT-IR cells in

single LSO segments along its tonotopic axis of adult (P40–P63) gerbils (n ¼ 5). The fraction of Ucn/ChAT neurons increases significantly toward

the most medial segments of the LSO. I: Percentage of Ucn/ChAT-IR cells relative to the total number of ChAT-IR cells for all lateral (L, L1–L5) and

all medial (M, M1–M5) limbs of the LSO of adult (P40–P63) gerbils (n ¼ 5). All error bars indicate standard errors of the mean. A magenta-green

copy is available as Supplementary Figure 1. For abbreviations, see list. Scale bar ¼ 200 lm in A–C (that in C also applies to E,F).



corrected for the average size of the cell nucleus by using

the Abercrombie correction factor (ratio of section thick-

ness divided by section thickness plus mean diameter of

nuclei). The diameter was estimated as the mean of 50 cell

nuclei of ChAT-IR LOC neurons with and without Ucn dou-

ble labeling. The mean diameter of the cell nuclei for both

types of LOC neurons was not different (8.4 lm) and

resulted in an Abercrombie correction factor of 0.8 (Aber-

crombie, 1946; Guillery, 2002).

Quantification of synapses in the innerspiral bundle of the cochlea

For quantification of the amount of Ucn-containing syn-

apses along the tonotopic axis of the inner spiral bundle

(ISB) underneath the inner hair cell region of the cochlea,

triple immunolabeling with antibodies directed against

Ucn, ChAT, and SV2 was applied (n ¼ 16, total number of

gerbils). As shown previously (Layton et al., 2005), SV2 is

absent from ribbon synapses of the afferent fibers contact-

ing the inner hair cells. Therefore all SV2-IR terminals in

the ISB region can be referred to as synaptic endings of

efferent fibers. Up to nine stacks of confocal images were

obtained (step size 0.3 lm) from the inner hair cell layer of

apical, middle, and basal parts of the cochlea and meas-

ured as distance from the apical end of the cochlea. SV2-,

Ucn/SV2-, ChAT/SV2-, and Ucn/ChAT/SV2-IR synaptic

endings were quantified within the whole depth of the

stack by using ImageJ plugins. Distances from the apical

end to the middle of the scanned cochlear areas were

determined on lower magnification images by measuring

the middle of the bleached background squares resulting

from the preceding confocal stack acquisition.

Data analysisResults from quantitative anatomical data are presented

as mean and standard error of the mean. For multiple sta-

tistical comparison of different brain areas or age groups

depending on whether the data passed the test for normal-

ity, Student’s t-test, one-way ANOVA, Kruskal-Wallis one-

way analysis of variance test (KW), or Mann-Whitney rank

sum test (MW) was performed. For comparison of different

trends in age groups we calculated the Pearson product

moment correlation. As a minimum criterion for statistical

significance, P < 0.05 was selected (Zar, 1974).

RESULTS

Urocortin-IR reveals a topographic gradientfor a stress-related subpopulation ofcholinergic neurons in the adult LSO

Consistent with previously published data from mouse

(Vetter et al., 2002; Wang et al., 2002) and rat (Bittencourt

et al., 1999; Wang et al., 2002; Gaszner et al., 2009), our

Ucn immunostaining of transversal sections from adult ger-

bil brainstem revealed typical Ucn-IR in the non-pregan-

glionic part of the Edinger-Westphal nucleus (Fig. 1A) and in

the LSO (Fig. 1C,D). The gerbil LSO has two anatomically

well-distinguished parts—the lateral and the medial limb.

The mediolateral axis of the LSO corresponds to a gradient

from low- to high-frequency projections (Sanes et al., 1989).

As can be seen in Figure 1C and D, Ucn-IR cells in adult ger-

bil are mainly located in the medial limb and are nearly

absent in the lateral limb of the LSO. Cholinergic cells within

the LSO cells belonging to the LOC system are distributed

throughout the entire volume of the LSO (Fig. 1C,D), with a

higher density, however, in the dorsomedial part of the LSO.

To compare the staining with typical cholinergic non-

auditory brain structures, immunostaining with anti-ChAT

antibodies is shown for the motoneurons of the facial nu-

cleus (Fig. 1B) in the adult gerbil, which is consistent with

the typical mammalian pattern (Motts et al., 2008). In the

superior olivary complex, ChAT-IR cells are located in and

around the LSO, and in most periolivary nuclei, especially

the ventral nucleus of the trapezoid body and the superior

paraolivary nucleus (data not shown).

Figure 2. Ucn-IR neurons of the LSO project to the cochlea and are of olivocochlear origin. Triple labeling of LSO neurons with the retrograde

tracer CTB from the cochlea and subsequent immunolabeling with anti-ChAT and anti-Ucn antibodies in the adult gerbil LSO. A: Overlay of CTB,

Ucn, and ChAT labeling. All retrogradely labeled LOC neurons (blue) were ChAT-IR (green). A subset of retrogradely labeled LOC neurons in the

medial limb of the LSO are also Ucn-IR (red). Dashed line shows the borders of the LSO. Maximum projection of several optical sections are

shown. B–D: Corresponding images of the same section as in A for CTB (B), Ucn (C), and ChAT (D) labeling only. All CTB-labeled cells are

stained for ChAT. E–H: Examples of neurons with higher magnification from the medial limb of the LSO showing maximum projection of several

optical sections. Corresponding labeling for Ucn-IR or CTB (E,F) and ChAT-IR or CTB (G,H), respectively. The neurons indicated by arrows were

double-labeled for Ucn and CTB (E,F) or ChAT and CTB (G,H). Note that in F a retrogradely labeled neuron is shown that does not show labeling

of Ucn-IR. All retrogradely labeled LSO neurons were double-labeled for ChAT. I: Percentage of retrogradely labeled CTB cells relative to either

Ucn-IR cells (depicted in black) or ChAT-IR cells (depicted in white) as shown for all lateral (L) and medial (M) LSO limbs in adult (P61–P63) ger-

bils (n ¼ 4). In the medial limb about 80% of ChAT-IR neurons or Ucn-IR neurons were double-labeled with CTB. In the lateral limb, also about

80% of ChAT-IR, but only 40% of Ucn-IR, neurons showed co-localization for CTB cells, reflecting the sparse amount of Ucn cells in the lateral

limb, as described in Figure 1. A magenta-green copy is available as Supplementary Figure 2. For abbreviations, see list. Scale bar ¼ 200 lmin A (applies to A–D); 40 lm in E (applies to E,F) and G (applies to G,H).

Kaiser et al.


To estimate the extent of co-localization of Ucn- and

ChAT-IR in different regions of the LSO along its tonotopic

axis, double-labeled cells were counted in the segments of

lateral (L1–L5; see Material and Methods) and medial limbs

(M1–M5) of the LSO (Fig. 1D). As can be seen in Figure 1D,

Ucn-IR cells are nearly absent from the lateral limb. Their

density, expressed in cells per square millimeter, increased

toward the medial limb of the LSO and reached the

Figure 2



maximum in the dorsal part (M4, M5) of the medial limb (Fig.

1G), whereas ChAT-IR cells were more evenly distributed

throughout the LSO (n¼ 4, P< 0.001, MW on linear regres-

sions). As all Ucn-positive cells were found to be cholinergic,

they clearly compose a subpopulation of LOC neurons.

When the number of Ucn-positive cells were expressed as

percentage of all cholinergic LOC neurons for each of the

segments (Fig. 1H), their non-uniform distribution was evi-

dent (n ¼ 5, P < 0.001, KW test, R2 ¼ 0.9803). In the adult

gerbil LSO, the percentage of Ucn-IR cells of all olivocochlear

neurons was on average about eightfold higher in the medial

compared with the lateral limbs (Fig. 1I, n¼ 5, P< 0.001, t-

test).

Ucn-IR cells of the LSO are olivocochlearneurons as demonstrated by retrogradetracing in adult gerbils

Following intracochlear injection of the fluorochrome-

coupled retrograde tracer CTB, we observed many la-

beled neurons in different parts of the superior olivary

complex. As expected, most labeled cells were found in

the ipsilateral LSO, whereas only a few weakly labeled

LOC neurons could be detected in the contralateral LSO.

In addition, neurons of the medial olivocochlear bundle

(MOC) were labeled ventrally in the proximity of the

medial superior olive (MSO), within the VNTB region, and

dorsal to the MSO. In this study, we limited our qualitative

and quantitative analysis to the LOC neurons in the ipsi-

lateral LSO. It is widely assumed that most or all LOC

cells are cholinergic, and ChAT is considered a marker for

cholinergic cells (German et al., 1985; Maley et al.,

1988). The visualization of retrogradely labeled LOC neu-

rons, ChAT-IR cells, and Ucn-IR cells plus their overlay

shows a coincident distribution of ChAT- and CTB-labeled

cells throughout the LSO, whereas Ucn-IR cells make up

only a part of the ChAT- and CTB-labeled cells (Fig. 2A–

D), as is obvious at higher magnification (Fig. 2E,G). Quan-

titative analysis of co-localization in lateral and medial

limbs showed that about 80% of cholinergic LSO neurons

and about 40–80% of Ucn-positive cells were labeled with

CTB (Fig. 2I, n ¼ 4, P < 0.05, ANOVA).

Expression of Ucn in cholinergic LOCneurons is age dependent

To investigate whether Ucn cells comprise a stable sub-

population of cholinergic LOC neurons during ontogeny, we

analyzed LSO sections from gerbils at different develop-

mental stages by using double immunolabeling with anti-

bodies against ChAT or Ucn (Fig. 3A–D). In general, the dis-

tribution and amount of cholinergic cells within the LSO of

P9, P14, P21–24, and P63 gerbils remained unchanged.

However, the expression of Ucn in P9–P63 animals was

subject to extensive changes. Before hearing onset, which

is at about P12 in gerbils (Woolf and Ryan, 1984), in P9 ani-

mals Ucn-IR cells were observed mainly in the medial limb

of the LSO and comprise approximately 10% (10.42 6

4.52) of ChAT-positive cells in the lateral and 64% (63.666

1.78) in the medial limb (Fig. 3A,E).

After hearing onset, however, in P14 animals the

expression of Ucn was significantly upregulated in both

limbs of the LSO. In these animals Ucn expression was so

strong that in addition to the somata, the cell processes

could clearly be identified, and the axons of the olivoco-

chlear bundle dorsally from the LSO (Fig. 3B) and

throughout their dorsal route to the eighth nerve were

visible (not shown here). At P14, Ucn-IR cells constituted

83–93% of ChAT-positive cells (L: 83.30 6 4.39; M:

93.486 2.46) in all LSO regions (Fig. 3E, n ¼ 4, compari-

sons different for all pairs except P9 vs. P40–63, P <

0.05, ANOVA). At P21–26, the amount of Ucn cells

decreased in both limbs, and in the lateral limb they com-

prised 40% of ChAT cells (39.65 6 10.13). In the medial

limb of P21–26 animals, the amount of Ucn-IR cells was

about 66% from cholinergic cells (66.61 6 6.28). After

P40 and up to P63, the LSO acquired an ‘‘adult’’ appear-

ance (n ¼ 4, multiple comparisons different for all pairs

except P21–26 vs. P9, P < 0.05, ANOVA). In the lateral

limb, about 4% of ChAT-IR cells were positively stained for

Ucn (3.58 6 1.97), whereas in the medial limb, the per-

centage of Ucn-IR from cholinergic cells remained similar

to that of the young gerbils, decreasing insignificantly to

about 40% (43.356 3.39).

All Ucn-IR LOC neurons co-express GABAand CGRP

As already mentioned above, all Ucn-IR cells of the LSO

showed co-localization for ChAT-IR (Fig. 4A). In order to eval-

uate whether LOC cells that were immunoreactive for Ucn

and ChAT also expressed GABA and CGRP, as described for

LOC cholinergic cells of other mammalian species (Safied-

dine et al., 1997; Raji-Kubba et al., 2002), immunostaining

of LSO sections with antibodies directed against GAD67,

GABA, and CGRP (Table 1) was performed. Analysis of these

sections revealed that throughout the range of age of gerbils

tested in this study, all Ucnþ/ChATþ cells as well as

Ucn�/ChATþ cells in the LSO appeared to contain GABA

and GAD67 (Fig. 4A–I). The immunoreactivity for GABA and

for GAD67 antibodies observed was relatively moderate and

comparable to that found in principal cells of the LSO. It is

worth mentioning that in both limbs of the LSO a small num-

ber of cells, obviously not the principal cells, exhibited

extremely high immunoreactivity for GAD67 and GABA

(data not shown). These cells, however, were not stained for

Ucn and/or ChAT immunoreactivity.

Kaiser et al.


Figure 3. Transient developmental changes of urocortin expression in LOC neurons of LSO. A–D: Maximum projection of several optical sections

obtained from the LSO labeled with anti-urocortin antibodies at postanatal age P9 (A), P14 (B), P24 (C), and P61 (D). Inverted depiction of mono-

chrome fluorescence images. Before hearing onset (P9, A) and in adults (P61, D), the pattern of distribution of Ucn-IR cells is similar, being mainly

concentrated in the medial limb. Immediately after hearing onset (P14, B), however, the Ucn-IR cells are more numerous, more intensely stained

(including dendrites and axons), and more evenly distributed throughout the LSO. This upregulation is still visible 12 days after hearing onset (P24,

C), but in an attenuated fashion. Note that in B and C dorsally to the LSO Ucn-IR axons are visible, which give rise to the LOC part of the olivocochlear

bundle. E: Percentage of Ucn-IR neurons relative to ChAT-IR cells for the lateral (L) and medial (M) limb of the LSO at postnatal day P9 (n ¼ 4), P14

(n ¼ 4), P21–24 (n ¼ 4), and P40–63 (n ¼ 5) calculated for a thickness of the LSO of 160 lm. In both limbs the overall ratio of Ucn-IR to ChAT-IR

cells is highest for P14 and P21–26 animals compared with P9 and P40–63 animals. This transitional upregulation of Ucn in LOC neurons is more

prominent in the lateral limb of the LSO, where Ucn-IR was almost absent in P9 and P40–63 gerbils. Scale bar ¼ 200 lm in A (applies to A–D).



Figure 4. Co-expression of GABA and CGRP in Ucn-IR neurons. A–L: Examples of LOC neurons showing co-localizations presented as overlay

(A,D,G,J), Ucn-IR (C,F,I,L), and immunoreactivity for ChaT (B), GABA (E), GAD67 (H), and CGRP (K), respectively. In A, D, G, and J, overlays for

triple staining with antibodies against MAP2 (green), Ucn (blue) plus (green) ChAT (A) or GABA (B) or GAD67 (C) or CGRP (D) are shown. Corre-

sponding single-channel images are shown in B, E, H, K, and C, F, I, and L as indicated. Arrows in yellow indicate the position of Ucn-IR neurons

for each series. All Ucn-IR LOC neurons showed co-expression of ChAT, GABA, GAD67, and CGRP. Single optical sections. A magenta-green

copy of this figure is available as Supplementary Figure 4. For abbreviations, see list. Scale bar ¼ 20 lm in A (applies to A–L).

Kaiser et al.


In contrast to MOC neurons of gerbil brainstem (data

not shown), we observed CGRP-IR in LOC neurons. Simi-

lar to recent findings in the hamster (Raji-Kubba et al.,

2002), the CGRP-positive cells in gerbils were always

located within the LSO, and staining was absent in neu-

rons of the shell region of the LSO, as can be seen in Fig-

ure 5B and D. For all age groups, the Ucn-IR cells always

showed co-localization with CGRP (Fig. 5A,C,E,F). The

density and proportion of Ucn-positive to Ucn-negative

cells throughout the lateral and medial limbs of the LSO

were generally comparable to that of cholinergic cells in

all age groups (n¼ 2, P< 0.05, KW). In Figure 5A–D, dou-

ble staining for Ucn/CGRP and Ucn/ChAT are compared

in adjacent transversal sections of the LSO for two age

groups. At P14, CGRP-, ChAT-, and Ucn-IR cells were

densely packed throughout the LSO, and Ucn-IR was

found in about 90–96% of CGRP and ChAT cells in both

limbs (Fig. 5E). During maturation, however, the density

of CGRP- and ChAT-IR cells in both limbs of the LSO

clearly decreased (Fig. 5F), as shown in adult gerbils

(P63), and Ucn-IR cells comprised about 8% in the lateral

and about 39–45% in the medial limb from CGRP as well

as ChAT cells (Fig. 5F).

Tonotopic distribution of Ucn-IR inolivocochlear terminals in the inner hair cellregion of the adult gerbil cochlea

Double immunostaining of cochlear wholemounts

against ChAT and Ucn clearly showed ChAT-positive ter-

minal arbors and putative synaptic endings of olivoco-

chlear efferents beneath the three rows of outer hair cells

and the one row of inner hair cells throughout the adult

gerbil cochlea (Fig. 6A–C). In agreement with Ucn staining

of terminals in the mouse cochlea (Vetter et al., 2002), in

the gerbil, immunoreactivity for Ucn was restricted to the

region of the inner spiral bundle (ISB) underlying the inner

hair cells (Fig. 6B,C). However, in contrast to the mouse,

in the adult gerbil Ucn-IR was confined to the middle and

basal part of the cochlea. The apical part of the bundle

(and other regions of the organ of Corti) were clearly

devoid of staining for Ucn (Fig. 6A). In accordance with

our findings in somata of the LSO, all Ucn-IR terminal

structures were double-labeled for ChAT.

To visualize the inner hair cells and olivocochlear Ucn-

positive terminals, double immunostaining for Ucn and cal-

retinin, which is a marker for inner hair cells and afferent

terminals (Dechesne et al., 1994), was performed and

revealed Ucn staining in the area where inner hair cells

were contacted by terminals of afferent nerve fibers (Fig.

6D–I). With higher magnification (Fig. 6G), this double im-

munostaining showed bouton-like Ucn-IR structures with a

diameter of about 1 lm at the very base of the inner hair

cells in very close relationship to contacting afferent fibers.

To analyze the nature of the Ucn-IR structures under-

neath the inner hair cells, we additionally stained the

cochlea with antibodies directed against SV2, a widely

used synaptic marker (Nealen, 2005). Double immuno-

staining with anti-Ucn/anti-SV2 antibodies demonstrated

that a significant part of the Ucn-positive structures

underneath the inner hair cells are SV2 positive (Fig. 6J–

L). Recently, it has been shown that SV2-IR is absent

from ribbon synapses, the special synapses of inner hair

cells (Layton et al., 2005). Thus, we conclude that the co-

localization of Ucn- and SV2-IR in the structures under-

neath the inner hair cells represents synaptic terminals of

olivocochlear efferents on spiral ganglion afferent termi-

nals. Confocal images taken perpendicular to the longitu-

dinal axis of the inner hair cells (Fig. 6J) revealed a ring-

like structure, which is composed of synaptic terminals

around the basal part of each inner hair cell.

Ucn-IR olivocochlear synapses show co-localization for CGRP and GAD65immunoreactivity

To compare the co-localizations for putative transmit-

ters in Ucn-IR olivocochlear cells of the LSO with their ter-

minals in the cochlea, we used triple immunostaining for

ChAT/Ucn/SV2. As can be seen in Figure 7A–D, Ucn-IR

presynaptic regions were always labeled with anti-ChAT

antibodies, which is consistent with the fact that Ucn-IR

cells within the LSO appeared to be of cholinergic nature

(Figs. 1C–F, 4A, 5B,D). Part of the presynaptic terminals

defined with anti-SV2 antibodies was IR for ChAT but not

for Ucn, and in some of the SV2-positive synapses, both

Ucn- and ChAT-IR was absent.

As we have already shown above, the somata of all

ChAT-IR (including Ucn-IR) olivocochlear neurons within the

LSO also demonstrated immunoreactivity for GABA and

GAD67 antibodies, suggesting that GABA is a co-transmit-

ter, which is consistent with earlier descriptions in other

mammalian species (Helfert et al., 1992; Henkel and

Brunso-Bechtold, 1998; Korada and Schwartz, 1999; Kan-

dler et al., 2002; Jenkins and Simmons, 2006). To further

investigate this co-localization, we carried out immunostain-

ing of the cochlea with antibodies directed against GAD65,

which is present in presynaptic terminals of GABAergic

cells (Kaufman et al., 1991; Soghomonian and Martin,

1998). The application of anti-GAD65 antibody within the

cochlea revealed an immunolabeling pattern similar to

staining with antibodies directed against SV2 (Fig. 7D,H,L).

Triple staining with ChAT/Ucn/GAD65 antibodies

demonstrated that within the ISB/inner hair cell region,

the GAD65-IR synaptic endings were either negative for

both ChAT and Ucn, or ChAT-positive and Ucn-negative,

or ChAT- and Ucn-positive (Fig. 7E–H). In addition, the



Figure 5. Colocalization of CGRP and Ucn in LOC neurons in young and adult gerbils. A–D: Overlays of double-stainings (A,C) for Ucn

(red) plus CGRP (green) and Ucn (red) plus ChAT (green) (B,D) for P14 (A,B) and P63 (C,D) animals. All the Ucn-IR neurons show double-

labeling for CGRP and ChAT. Note that the comparably large shell LOC neurons in B and D do not show co-expression for Ucn or CGRP.

E,F: Percentage of Ucn-IR cells relative to ChAT-IR cells (black bars) and CGRP-IR cells (white bars), within the medial (M) and lateral (L)

limbs of the LSO in P14 (E, n ¼ 2) and in P63 (F, n ¼ 2) gerbils, respectively. Shortly after hearing onset, in P14 animals almost all cells

showed co-localization for Ucn, ChAT, and CGRP. In adults (P63), however, the overall number and the mediolateral distribution Ucn-IR

neurons changed significantly. The magnitudes of all changes in number of Ucn-IR neurons in respect to the co-localization with ChAT and

CGRP were in parallel. A magenta-green copy is available as Supplementary Figure 5. For abbreviations, see list. Scale bar ¼ 200 lm in A

(applies to A–D).

Kaiser et al.


pattern of immunolabeling in the inner hair cell region of

the cochlea for CGRP strongly resembled that observed

for ChAT-IR (Fig. 7I–L). Again, staining with SV2 antibod-

ies demonstrated three types of transmitter combinations

in the presynaptic terminals: 1) exclusive immunostaining

for SV2; 2) co-localization for SV2 and CGRP; and 3) tri-

ple-IR for SV2/Ucn/CGRP. Similar to the ChAT staining

pattern and in contrast to the Ucn staining, GAD65- and

CGRP-IR was observed throughout the whole length of

the cochlea (not shown).

Figure 6 (legend on page 2772).



The frequency-specific distribution of Ucn-IRin olivochochlear terminals changes withcochlear development

In contrast to all other putative transmitters investigated

here (Fig. 6), the distribution of Ucn-IR was not homoge-

nous throughout the adult gerbil cochlea, exhibiting a dis-

tinct lack of Ucn innervation in the apical part. To investi-

gate whether this adult pattern is a stable feature during

postnatal development, we analyzed Ucn immunolabeling

of apical, middle, and basal cochlear turns in four different

age groups (Fig. 8), i.e., 1) before hearing onset (P9); 2)

shortly after hearing onset (P14); 3) adolescent (P21–26);

and 4) adult gerbils (P61–63). Before the onset of hearing

(P9), which in gerbils typically occurs at around P12 (Woolf

and Ryan, 1984), the apical, low-frequency part of the coch-

lea was devoid of Ucn labeling (Fig. 8A) and in the middle

part a very faint fluorescent signal was observed in the ISB

area (Fig. B). Only in the basal, high-frequency part of the

cochlea single, were approximately 1-lm-sized structures

brightly labeled (Fig. C). After hearing onset (P14) and up to

P21–26, a bright Ucn-fluorescent signal was observed

throughout the whole cochlear extent (Fig. 8D–I). However,

this more or less homogenous distribution of Ucn-IR was

not stable during maturation of the animals. Beginning with

P40, the staining disappeared from the apical region,

decreased in the middle part, and remained brightly visible

in the basal part of the cochlea (Fig. 8J–L).

Transient developmental changes in Ucnexpression in LSO somata and cochlearterminals of olivocochlear neurons

To quantify and compare the postnatal developmental

changes in Ucn-IR of olivocochlear neurons in the LSO

and their efferent terminals on the afferent dendrites of

spiral ganglion cells underneath the inner hair cells of the

cochlea, we enumerated the ChAT- and Ucn/ChAT-posi-

tive synapses at different sites along the tonotopic axis of

cochlea for all age groups studied (Fig. 9). In Figure 9H

the frequency presentation of the adult gerbil cochlea is

included in the top axis (data calculated from Muller,

1996). Our analysis confirmed the qualitative findings

from Figure 8. In both the LSO (n ¼ 4) and the cochlea,

Ucn-IR was restricted to high-frequency regions before

hearing onset (P9, Fig. 9A,E; LSO: R ¼ 0.975, P <

0.000001; cochlea: R ¼ 0.878, P < 0.0002), was then

upregulated after hearing onset (P14–26, Fig. 9B,F; LSO:

R ¼ �0.192, n.s.; cochlea: R ¼ 0.150, n.s.; Fig. 9C,G;

LSO: 0.713, P < 0.02; cochlea: R ¼ �0.405; n.s.)

throughout the cochlea, and eventually decreased in

adult animals (P40–63, Fig. 9D–H; LSO: R ¼ 0.932, P <

0.0001; cochlea: R ¼ 0.639, P < 0.001), almost disap-

pearing from the low-frequency part of the cochlea (n ¼ 2

for P9 and P21–26, n¼ 3 for P14 and P60–63).

DISCUSSION

In contrast to most other laboratory animals, gerbils

have a hearing range that extends to low frequencies, as

in humans. Therefore we investigated the urocortinergic

neurons in the lateral olivocochlear system of adult and

developing gerbils. The results of our study show several

new findings, which are important for understanding the

role of urocortin and stress in the peripheral auditory

pathway. First, we demonstrate for the first time a direct

neuroanatomical projection of Ucn-IR LOC neurons to the

inner hair cell region of the cochlea. Second, in the adult

gerbil we find a clear tonotopic gradient of Ucn-IR cells in

Figure 6. Distribution of cholinergic and urocortinergic olivocochlear axons and synaptic terminals along the topographic axis of the adult gerbil

cochlea. A–C: Cochlear efferent fibers and synapses in apical (A), mid-apical (B), and basal (C) regions of cochlea; double staining for urocortin

(depicted in red) and ChAT (depicted in green). ChAT-IR MOC fibers cross the tunnel of Corti (TC) and terminate with large synapses at the base of

the three rows of outer hair cells. At the bottom, the inner spiral bundle (ISB) underneath the one row of inner hair cells is visible as a thin strip of

ChAT-IR. The terminals of these LOC fibers are smaller and can be seen as puncta of Ucn-IR (red) and/or ChAT-IR (green). The pattern of ChAT

staining (green) for MOC and LOC fibers is similar in all three regions. Ucn-IR (red), however, is restricted to the ISB, where LOC fibers terminate on

afferent dendrites, which synapse on inner hair cells. Note that Ucn-IR is unevenly distributed along the longitudinal extent of the cochlea. Ucn-IR is

absent in the apical part (A), moderate in the mid-apical region (B), and maximal in the basal part of cochlea (C) of the adult gerbil cochlea. D–F:

Detail of the basal part of the cochlea showing the inner hair cells. Note that compared with A–C, the viewing angle is tilted by about 90�. D: Over-

lay. E,F: Corresponding single-channel images for Ucn-IR and calretinin-IR as indicated. Inner hair cells (IHCs) and contacting radial afferent fibers

(a) are stained with anti-calretinin antibodies (green) as shown in D and E. Close to the base of the IHCs, a band of Ucn-IR fibers and terminals (red)

can be distinguished that comprises the ISB.G–I: Same part of the inner hair cell region at higher magnification. Note well-distinguishable contacts

(asterisks) of afferent fibers (a) with inner hair cells (IHC) as marked with anti-calretinin (G,I). Ucn-containing efferent fiber endings (arrows) lie in

close proximity to the afferents. Color coding as in D–F. J–L: Inner hair cell region of the basal part of the cochlea viewed in the longitudinal axis of

the inner hair cells as in A–C and focused in the plane of Ucn-IR terminals. Double staining with anti-Ucn (red) and SV2 (green) antibodies (overlay

in J; corresponding monochrome channels for Ucn in K and SV2 in L) reveals a ring-like structure composed of Ucn-IR efferent terminals (green)

including synapses (vesicular marker SV2 in red) in close proximity to the base of the hair cells. A–F, maximum projection of several single optical

sections; G–L, single optical confocal sections. A magenta-green copy of this figure is available as Supplementary Figure 6. For abbreviations, see

list. Scale bar ¼ 40 lm in C (applies to A–C); 20 lm in D (applies to D–F); 5 lm in G (applies to G–I); 10 lm in J (applies to J–L).

Kaiser et al.


the LSO and their terminals in the cochlea, with a lack of

expression in the low-frequency regions. Third, we show

that Ucn-IR neurons comprise a subpopulation of LOC

neurons and show co-localization for ChAT, CGRP, and

GABA. Finally, we demonstrate a transient upregulation

of Ucn in LOC neurons after hearing onset.

From previous retrograde tracing studies it was evident

that two populations of cholinergic neurons (Warr, 1975;

Altschuler et al., 1985; Vetter et al., 1991) within and

near the LSO project to the cochlear epithelium. Bitten-

court and colleagues (1999) showed that in the mamma-

lian brain many different nuclei contain neurons that

express Ucn. However, only one of them, the LSO,

belongs to the auditory system (Bittencourt et al., 1999).

Subsequently, in the cochlea of mice an extensive net-

work of Ucn-IR fibers in the inner spiral bundle and pre-

sumably in synaptic terminals in close proximity to inner

hair cells has been shown (Vetter et al., 2002). Although

these studies suggested that Ucn was a co-transmitter of

LOC neurons, direct proof was missing. By using retro-

grade tracing of fluorochrome-coupled CTB and immuno-

histochemistry against Ucn and other antibodies in adult

gerbils (Fig. 2), we were able to demonstrate that Ucn-

LOC neurons send their axons to the inner spiral bundle

of the cochlea and terminate with their synapses on the

afferent dendrites of auditory afferents (Figs. 6, 7).

Our detailed analysis of the distribution of Ucn neurons

in the LSO and their fibers and terminals in the cochlea

revealed a topographic gradient that has not been

described before. However, a prerequisite for the discov-

ery of this gradient was the use of species with an

extended low-frequency hearing. Other rodents, like mice

and rats, lack low-frequency hearing and therefore are

not suitable for investigating topographic gradients in

Figure 7. Co-localization of CGRP and GAD65 in Ucn-IR cochlear efferent synapses. A–L: Single optical confocal sections obtained from

the middle part of the adult (P63) cochlea at the base of inner hair cells. Triple staining with antibodies against ChAT/Ucn/SV2 (A–D),

Ucn/ChAT/GAD65 (E–H), or Ucn/CGRP/SV2 (I–L), respectively. Overlays and corresponding single-channel images are shown as indi-

cated. A–D: Overlay (A) of Ucn (red), ChAT (green), and the vesicle marker SV2 (blue) and the corresponding single-channel images (B–D)

reveal a stripe of synapses including ring-like structures. Almost all synaptic endings showed co-localization for ChAT-IR. In addition, a frac-

tion of the SV2/ChAT terminals were also Ucn positive (arrowheads). E–H: Overlay (E) and corresponding monochrome images (F–H) for

Ucn (red), ChAT (green), and GAD65 (blue) immunoreactivity. The overall labeling pattern was similar to that of SV2 (compare D and H),

reflecting distribution of GABAergic synapses. Almost all GAD65-positive synaptic endings were also ChAT-IR. Again, as in A, some of the

GAD65/ChAT-positive synapses were triple-labeled for Ucn (arrowheads). I–L: Overlay (I) and single-channel images (J–L) for Ucn (red),

CGRP (green), and SV2-IR (blue). The anti-CGRP staining pattern (I,K) was similar to that of ChAT (C,G). A fraction of SV2/ChAT-IR termi-

nals were also Ucn-positive (arrowheads). A magenta-green copy of this figure is available as Supplementary Figure 7. For abbreviations,

see list. Scale bar ¼ 10 lm in A (applies to A–L).



that range. The results from our study suggest that Ucn

LOC neurons in adult gerbils are capable of modulating

spontaneous and tone-evoked activity of the dendrites of

auditory afferents in middle and high frequencies, but not

in the low-frequency range. Whether there is a similar dis-

tribution and putative effect in humans is not known. In

Figure 8. Developmental changes in Ucn-IR of cochlear efferent synaptic terminals along the cochlear tonotopic axis. Single optical confocal

sections acquired from apical (A,D,G,J), middle (B,E,H,K), and basal parts (C,F,I,L) of cochleas from four age groups (P9–P63) reveal dynamic

changes in the distribution of Ucn-IR. Locations of samples relative to the apical end along the cochlea were 100–500 lm (apical), 500–2,000

lm (medial,) and 2,000–10,000 lm (basal).A–C: In P9 gerbil Ucn-IR varies from not detectable (apical A), faint (middle B), to moderate in the

basal part of the cochlea (C). D–I: Ucn staining in P14 (D–F) and P24 animals (G–I) results in a comparably bright labeling, for all parts of the

cochlea. The overall intensity of labeling was slightly more intense in P14 cochleae than in P24. J–L: In adult gerbil cochleae (P63) the apical

part was devoid of Ucn (J), the middle part was moderately stained (K), and the basal part showed the most intense Ucn-IR (L). A magenta-

green copy of this figure is available as Supplementary Figure 8. Scale bar ¼ 10 lm in A (applies to A–L).

Kaiser et al.


Figure 9. Transient developmental changes of Ucn-IR in the LSO and the cochlea. A–D: Change of percentage of Ucn-IR neurons relative to

ChAT-IR neurons in the LSO as expressed for lateral (L1–L4) and medial (M1–M5) segments of the LSO for P9 (A), P14 (B), P21–26 (C), and

P60–63 (D) animals (n ¼ 4 for all age groups). Note the similar distributions in pre-hearing (A) and adult (D), and the transient upregulation of

Ucn-IR cells in the juvenile stages (B,D). E,F: Percentage of Ucn-IR synapses relative to all ChAT-IR synapses underneath the inner hair cells of

the cochlea along the tonotopic gradient from apical to basal during development. G,H: In H (top axis), the corresponding frequency representa-

tion for the adult cochlea is included. In the cochlea, the Ucn-IR is restricted to middle and high-frequency parts in P9 (E) and adult (H) animals.

In juvenile stages (F,G), Ucn expression is upregulated in the apical part of the cochlea. Note the clear match of urocortin expression patterns

in the LSO and cochlea for the different ages (P9, n ¼ 2; P14, n ¼ 3; P21–26, n ¼ 2; P60–63, n ¼ 3). In all panels R is calculated as the

Pearson correlation coefficient.


clinical studies it has been shown that different kinds of

stress (such as overall stress, emotional stress, and post-

traumatic stress disorder) can intensify sudden hearing

loss or tinnitus in humans (Ban and Jin, 2006; Hinton

et al., 2006; Fagelson, 2007; Hebert and Lupien, 2007; Al

Mana et al., 2008); in general, however, the affected fre-

quency range was not the focus of these studies. Tinnitus

occurs mostly as high-pitch, but can also occur as low-

pitch, ringing.

Another finding in our study was the lack of Ucn-IR in

shell neurons. Intrinsic and shell neurons of the LOC sys-

tem show a different innervation pattern in the cochlea.

Whereas shell neurons innervate larger parts of the coch-

lea with a corresponding frequency span of about one

octave, the innervated region in the cochlea of intrinsic

LOC neurons is much more restricted and covers about

0.2 octaves (Warr et al., 1997). Therefore we suggest that

Ucn-IR intrinsic LOC neurons are capable of modulating

auditory processing in afferents in a frequency-specific

manner. Such fine tuning of a putative stress-induced

modulation in the cochlea could, in theory, produce such

narrow-band effects as ringing in tinnitus or hearing loss

in certain frequencies. However, this in turn would also

require that under physiological conditions only a few and

not all Ucn-LOC neurons are activated.

From previous studies it is known that the LOC effer-

ents express (in addition to their main transmitter, acetyl-

choline) a variety of co-transmitters such as dynorphin,

CGRP, enkephalin, dopamine, GABA (Vetter et al., 2002;

Raphael and Altschuler, 2003), and Ucn (Vetter et al.,

2002). However, there was no information available on

which of these co-transmitters are simultaneously

expressed in Ucn-IR LOC neurons. Our analysis of the co-

expression of Ucn and acetylcholine, GABA/GAD65/

GAD67, and CGRP in the LSO and the cochlea (Figs. 4–7)

showed a co-localization of Ucn with all of these

neurotransmitters.

In conclusion, the LOC neurons provide a cocktail of

many different neurotransmitters at the afferent den-

drites underneath the inner hair cells. Whereas GABA and

acetylcholine were present throughout the LSO and coch-

lea, Ucn-IR was almost absent in the low-frequency

regions. Taking into account these findings, we suggest

that Ucn neurons make up a special subpopulation of

LOC neurons and that therefore there exist at least two

populations of LOC neurons as evaluated by the composi-

tion of their synaptic cocktail. Why these neurons use a

comparably large number of neurotransmitters, is, how-

ever, unclear.

Another major finding of our study was the develop-

mental changes in Ucn expression and distribution in neu-

rons of the LSO and their fibers in the cochlea. When we

compared pre-hearing animals with post-hearing and

adult animals, a dramatic transient upregulation of Ucn

expression was evident for animals immediately after

hearing onset, resulting in Ucn-IR not only in the high- but

also in the low-frequency regions of the LSO and cochlea

(Figs. 3, 5, 8, 9). These post-hearing changes in LOC neu-

rons are particularly interesting, as it is has been argued

that the cochlear and auditory brainstem circuitry is com-

plete before hearing onset (Simmons, 2002; Kandler

et al., 2009). The cause and function of this phenomenon

is unclear, but we speculate that the onset of hearing is a

transient physiological challenge for many parts of the

auditory system, which involves dramatic metabolic

changes due to auditory input and modulatory input from

other areas of the central nervous system.

Recently, Darrow and colleagues (2006) investigated

the putative functional significance of the LOC neurons

and suggested that lateral olivocochlear neurons are

involved in medium-term and long-term (tens of minutes)

balancing of interaural intensity differences. However,

why and how that would relate to the stress system

remains obscure.

Considering the challenges for the auditory system and

in particular the cochlea and auditory brainstem nuclei af-

ter hearing onset, the upregulation of Ucn expression in

LOC neurons could be causally linked to a balancing func-

tion of those neurons. For example, shortly after hearing

onset there could be a short period of time (days) that is

required for finally adjusting and balancing several inputs,

especially binaural ones. Interestingly, adult thresholds of

auditory brainstem responses (ABRs) in gerbils are not

complete at P30 (Woolf and Ryan, 1984), which is con-

sistent with our observed upregulation of Ucn in LOC

neurons until at least P26 (Figs. 3, 9). The mother-pup

vocalizations, however, occur almost exclusively in the ul-

trasonic range (Yapa, 1994), which could explain an up-

regulation of urocortin in the high-frequency part of the

cochlea. Such a putative behavioral significance of the

stress system could also be explained by a protective

effect of Ucn, which in general seems to be its role

regarding the homeostatic regulation of stress responses.

Urocortin seems to have an anxiolytic effect with a termi-

nation slow component compared with the initial fast

component at the beginning of the behavioral stress

response (Heinrichs and Koob, 2004; De Kloet et al.,

2005). Whether urocortin has such a protective role in

the cochlea is speculative and should be investigated by

physiological approaches.

ACKNOWLEDGMENTS

The monoclonal mouse SV2 antibody developed by

Kathleen M. Buckley was obtained from the Developmen-

tal Studies Hybridoma Bank, developed under the

Kaiser et al.


auspices of the National Institute of Child Health and

Human Development and maintained by The University of

Iowa (Department of Biological Sciences, Iowa City, IA

52242). We thank Beate Stiening and Hilde Wohlfrom for

performing Western blot analysis of gerbil brain lysate to

test the specificity of primary antibodies. The authors

thank Mario Wullimann for comments on the manuscript.

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