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Brain Structure and Function ISSN 1863-2653 Brain Struct FunctDOI 10.1007/s00429-013-0585-8

A fast cholinergic modulation of theprimary acoustic startle circuit in rats

Ricardo Gómez-Nieto, Donal G. Sinex,José de Anchieta C. Horta-Júnior,Orlando Castellano, Javier M. Herrero-Turrión, et al.

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ORIGINAL ARTICLE

A fast cholinergic modulation of the primary acoustic startlecircuit in rats

Ricardo Gomez-Nieto • Donal G. Sinex •

Jose de Anchieta C. Horta-Junior • Orlando Castellano •

Javier M. Herrero-Turrion • Dolores E. Lopez

Received: 15 February 2013 / Accepted: 16 May 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract Cochlear root neurons (CRNs) are the first

brainstem neurons which initiate and participate in the full

expression of the acoustic startle reflex. Although it has

been suggested that a cholinergic pathway from the ventral

nucleus of the trapezoid body (VNTB) conveys auditory

prepulses to the CRNs, the neuronal origin of the VNTB–

CRNs projection and the role it may play in the cochlear

root nucleus remain uncertain. To determine the VNTB

neuronal type which projects to CRNs, we performed tract-

tracing experiments combined with mechanical lesions,

and morphometric analyses. Our results indicate that a

subpopulation of non-olivocochlear neurons projects

directly and bilaterally to CRNs via the trapezoid body. We

also performed a gene expression analysis of muscarinic

and nicotinic receptors which indicates that CRNs contain

a cholinergic receptor profile sufficient to mediate the

modulation of CRN responses. Consequently, we investi-

gated the effects of auditory prepulses on the neuronal

activity of CRNs using extracellular recordings in vivo.

Our results show that CRN responses are strongly inhibited

by auditory prepulses. Unlike other neurons of the cochlear

nucleus, the CRNs exhibited inhibition that depended on

parameters of the auditory prepulse such as intensity and

interstimulus interval, showing their strongest inhibition at

short interstimulus intervals. In sum, our study supports the

idea that CRNs are involved in the auditory prepulse

inhibition of the acoustic startle reflex, and confirms the

existence of multiple cholinergic pathways that modulate

the primary acoustic startle circuit.

Keywords Biotinylated dextran amine � Cochlear root

neurons � Extracellular recordings � Olivocochlear bundle �Prepulse inhibition � Ventral nucleus of the trapezoid body

Introduction

In rodents, cochlear root neurons (CRNs) have large cell

bodies and thick dendrites oriented parallel or perpendic-

ular to the eighth nerve fibers among which they are dis-

tributed (Merchan et al. 1988; Lopez et al. 1993). They are

recipients of numerous cholinergic inputs from the ventral

nucleus of the trapezoid body (VNTB) (Gomez-Nieto et al.

2008a), converging glutamatergic inputs from auditory

nerve fibers (Harrison et al. 1962; Merchan et al. 1988;

Gomez-Nieto et al. 2008b), noradrenergic inputs from

the locus coeruleus (Gomez-Nieto et al. 2008b), as well

as GABA and glycinergic inputs of unknown origin

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00429-013-0585-8) contains supplementarymaterial, which is available to authorized users.

R. Gomez-Nieto � J. A. C. Horta-Junior � O. Castellano �J. M. Herrero-Turrion � D. E. Lopez

Neuroscience Institute of Castilla y Leon (INCyL), University of

Salamanca, 37007 Salamanca, Spain

R. Gomez-Nieto � O. Castellano � D. E. Lopez (&)

Department of Cell Biology and Pathology, Medical School,

University of Salamanca, 37007 Salamanca, Spain

e-mail: lopezde@usal.es

R. Gomez-Nieto � O. Castellano � D. E. Lopez

Institute of Biomedical Research of Salamanca (IBSAL),

University of Salamanca, 37007 Salamanca, Spain

D. G. Sinex

Departments of Psychology and Biology, Utah State University,

Logan, UT 84322-2810, USA

J. A. C. Horta-Junior

Department of Anatomy, Biosciences Institute, Sao Paulo State

University (UNESP), Botucatu, Sao Paulo 18618-970, Brazil

123

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DOI 10.1007/s00429-013-0585-8

Author's personal copy

(Osen et al. 1991; Gomez-Nieto et al. 2008b). The primary

neuronal circuit underlying the mediation of acoustic

startle reflex encompasses three central synaptic relay sta-

tions: CRNs, giant PnC neurons, and cranial and spinal

motoneurons (Lingenhohl and Friauf 1994; Lee et al. 1996;

Lopez et al. 1999; Nodal and Lopez 2003). Thus, CRNs are

the first brainstem neurons which initiate and participate in

the full expression of the acoustic startle reflex (Lopez

et al. 1999; Horta-Junior et al. 2008). CRNs contain all the

necessary neurotransmitter receptors to provide short

latency acoustic input to neurons in the caudal pontine

reticular nucleus (PnC); mediating alert and escape

behaviors elicited by a sudden and intense acoustic stim-

ulus (Lee et al. 1996; Lopez et al. 1999; Sinex et al. 2001;

Gomez-Nieto et al. 2008b). The acoustic startle response

can be reduced when a strong acoustic startling stimulus

(pulse) is shortly preceded by a weaker sound (prepulse), a

paradigm called auditory prepulse inhibition (PPI). Inves-

tigation of PPI mediating pathways is critical for estab-

lishing new animal models for studying both cognitive

features and neural bases of some neuropsychiatric disor-

ders, which are characterized by PPI deficits (Swerdlow

and Geyer 1998; Swerdlow et al. 2001; Li et al. 2009;

Molina et al. 2009). It is widely accepted that the inferior

colliculus (IC) relays auditory information to the primary

acoustic startle circuit at the level of the PnC (Leitner and

Cohen 1985; Li et al. 1998a, b; Fendt et al. 2001; Yeomans

et al. 2006), through a slow multimodal pathway which

serially connects the IC, the superior colliculus, and the

inhibitory cholinergic projection from the pedunculopon-

tine tegmental nucleus to the PnC (Koch et al. 1993; Fendt

and Koch 1999; Fendt et al. 2001; Li et al. 2009). Never-

theless, this neuronal circuit does not explain all of the

singularities of PPI such as the effectiveness of interstim-

ulus intervals as short as 20 ms (Hoffman and Ison 1980).

This leads us to presume the existence of another short

auditory projection to the primary acoustic startle circuit.

Previous studies suggested that the descending auditory

pathway IC–VNTB–CRNs conveys auditory prepulses at

the level of the cochlear root nucleus to mediate the

auditory PPI (Gomez-Nieto et al. 2008a). However, the

VNTB neuronal type which projects to the CRNs is not

known and no electrophysiological data is available to

support the inhibition of CRNs by auditory prepulses. This

study focuses on determining the neuronal type that is the

source of the cholinergic projection to the cochlear root

nucleus. In agreement with our morphological findings

(current study, Gomez-Nieto et al. 2008a, b), we also show

a gene expression analysis of cholinergic receptors and

electrophysiological data which supports that CRNs par-

ticipate in the auditory PPI of the acoustic startle reflex via

the VNTB–CRNs projection.

Materials and methods

Experimental animals

In total, 21 adult female Sprague–Dawley rats (Charles

River Laboratories) weighing 290–320 g were used in this

study. The experiments were conducted according to the

guidelines for the use and care of laboratory animals of the

European Communities Council Directive (DOCE L 222;

24-08-1999) and with those established by the United

States (NIH publication No. 80–23). All efforts were made

to minimize the number of animals used and their suffer-

ing. For the surgical procedures, the animals were deeply

anesthetized with a mixture of ketamine (40 mg/kg body

weight) and xylazine (7 mg/kg body weight), and main-

tained in this state by supplementary doses, as required,

throughout the duration of the experiment.

Neuroanatomical experiments: surgery, tissue

processing and image analysis

A total of 9 animals were used to study the source of the

VNTB inputs on the CRNs. The rats received unilaterally

injections of the bidirectional tracer, biotinylated dextran

amine (BDA, 10,000 MW; #D-1956; Molecular Probes,

Eugene, OR) into brain structures that are well known to

contain axonal tracts of VNTB neurons (bundles) or its

efferent innervations (Warr and Beck 1996). Specifically,

the tracer experiments included BDA injections into the

following areas: the VNTB, the medial olivocochlear

bundle (OCB), the ventral cochlear nucleus (VCN), and the

trapezoid body (TB). All surgical and stereotaxic proce-

dures for injecting the tracer were identical to that used in

our previous studies (Gomez-Nieto et al. 2008a, b; Horta-

Junior et al. 2008). BDA (10 % in distilled water) was

injected iontophoretically via a glass micropipette (25 lm

tip diameter), with 3 lA positive current pulses (7 s on/7 s

off) for a period of 10 min. The coordinates for the left

VNTB were precisely the same as those devised by

Gomez-Nieto et al. (2008a). The coordinates for the medial

OCB, the TB and the VCN were obtained from the atlas of

the rat brain (Paxinos and Watson 1998), using an electrode

angle calibrator (David Kopf Instruments). In another set of

experiments, two animals were injected with BDA into the

VNTB followed by a mechanical lesion of the OCB. The

lesion was made with a surgical needle which was aimed

with the stereotaxic apparatus to target the medial OCB.

The lesion was attempted contralateral to the injection site,

and placed off of midline at a point where olivocochlear

fibers travel. The needle was moved several times along the

rostro-caudal plane to ensure the surgical transection of

medial olivocochlear fibers.

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Tissue preparation for histology including perfusion of

the animals, Nissl staining, visualization of the BDA neu-

rotracer, and calbindin protein-D28 K (CaBP) immuno-

histochemistry was identical to those used in our previous

studies (Lopez et al. 1993, 1999; Gomez-Nieto et al.

2008a). Nickel-intensified peroxidase reaction was devel-

oped for the BDA visualization to distinguish from the

CaBP immunohistochemistry (Hancock 1982; Gomez-Ni-

eto et al. 2008a). Cochleae of animals with BDA injections

were also processed for the tracer visualization and were

used to analyze the distribution of medial olivocochlear

fibers in the cochlea, as in our previous study (Gomez-

Nieto et al. 2008a).

All sections were examined on an upright brightfield

microscope (#BX5; Olympus, Center Valley, PA, USA)

equipped with a digital camera (SpotRt�; Diagnostic

Instruments, Sterling Heights, MI, USA). Low-magnifica-

tion images were taken with the 49, 109 or 209 objective

lens, and high magnification images were taken with a 409

or 1009 objective lens (oil immersion) for morphometric

analysis of BDA-labeled structures.

The morphometric analysis of labeled structures was

carried out with ImageJ (version 1.42; Rasband, N.S.,

National Institutes of Health, Bethesda, Maryland, USA;

http://rsb.info.nih.gov/ij). Photomicrographs shown in the

figures were processed with minor modifications in

brightness, contrast and to remove the tissue-free back-

ground using Adobe Photoshop� (version 9.0; Adobe

Systems Incorporated, San Jose, CA, USA) and assembled

in Canvas 7.0 software.

Isolation of RNA and RT-PCR

The cochlear nerve roots of 8 rats were collected bilaterally

to study the gene expression of acetylcholine receptors,

particularly the muscarinic (M1-5) and nicotinic (Na4,

Na7, Nb2 and Nb3) receptors subunits. Prefrontal cortex

(PFC) and hippocampus (H) were also sampled to serve as

positive controls. Isolation of RNA and reverse transcrip-

tion-polymerase chain reaction (RT-PCR) were then per-

formed by standard procedures as described elsewhere

(Gomez-Nieto et al. 2008b). Total RNA was immediately

isolated from the individual tissue samples using the

TRIZOL� reagent (Gibco BRL, Gaithersburg, MD, USA)

in accordance with the manufacturer’s procedure. The

quantity of purified RNA was assessed by spectropho-

tometry Nano Photometer (Implen GmbH) and the integ-

rity subsequently confirmed with RNA 6000 Nano

LabChip (Agilent Technologies, Palo Alto, CA, USA),

obtaining the RNA integrity number (RIN, 0 corresponding

to fully degraded RNA and 10 corresponding to intact

RNA). For all PCRs, only RNA samples with RIN of at

least 7.5 were used, with the vast majority of samples

having a RIN of at least 8.0.

Total RNA (2 lg), primed with oligo-dT, was reverse-

transcribed into complementary DNA (cDNA) at 37 �C for

2 h using the first-strand cDNA synthesis kit (Promega

Corporation, Madison, WI, USA) in a 20 ll volume and

stored at -20 �C until use, according to manufacturer’s

instructions. In all cases, a reverse transcriptase negative

control was used for testing genomic DNA contamination.

Also, DNase treatment (Turbo DNA-free Kit, Applied

Biosystems) was performed at 37 �C for 2 h to remove

DNA contamination, followed by inactivation of the

DNases at 75 �C for 15 min.

A 25 ll PCR mixture contained 100 ng of cDNA tem-

plate, 20 pmol of each primer, 0.2 mM dNTPs, 1.5 mM

MgCl2 and 5 units of GoTaq Flexi DNA polymerase

(Promega Corp.). Supplemental figure 1 lists primers spe-

cific for the genes examined in the present study. PCR

reactions were performed in an ABI7000 PCR system

(Applied Biosystems, Europe). Amplification of rat b-actin

(GenBank accession no. NM_031144) was used as an

internal and loading control. PCRs amplification were as

follows: 1 cycle at 95 �C for 5 min as an initial denatur-

ation step, denaturation at 95 �C for 30 s, annealing at

57–59 �C for 30 s, and extension at 72 �C for 45 s (30

cycles), followed by further incubation for 10 min at 72 �C

(1 cycle). PCR conditions were shown to be at the linear

phase of amplification to assess a semiquantitative analysis.

PCR products were electrophoresed on 2.5 % agarose gels

in 1 9 40 mM Tris–acetate, 1 mM ethylenediamine tetra-

acetic acid pH 8.0, and visualized by ethidium bromide

staining.

The primers were designed in such a way that RT-PCR

products spanned two identified introns. Moreover, the

amplification of b-actin was performed with equal amounts

of RNA and demonstrated an identical expression pattern

in all cerebral regions used. Finally, in all RT-PCRs, an

RNA-free (negative) control sample was used which did

not produce any amplified bands.

Electrophysiology experiments: acoustic stimulation,

data collection and analysis

Four rats were used for the extracellular recording of CRNs

neuronal activity after auditory prepulse stimulation. The

rats were mounted in a stereotaxic apparatus using hollow

ear bars coupled to an earphone (#1310B, Radio Shack

Super Tweeter, Korea) for stimuli delivery. The recording

microelectrode was aimed to pass through the rat’s

cochlear root nucleus, using identical coordinates and

surgical procedures to those used in our previous studies

(Lopez et al. 1999; Sinex et al. 2001; Nodal and Lopez

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2003; Gomez-Nieto et al. 2008a; Horta-Junior et al. 2008).

Acoustic stimulation was performed using almost identical

devices and method to those used by Sinex et al. (2001).

The frequency response of the acoustic system was mea-

sured in a coupler with a calibrated microphone (#4136,

B&K, GA, USA). This standard calibration curve was used

to set the levels of tones in all experiments. To record the

neuronal activity, we used carbon fiber electrodes (#E1011,

Carbostar-1, Kation Scientific, MN, USA) with impedance

at 1 kHz of 0.4–0.8 MX and tip length of 15 lm. The

electrode was fixed to the stereotaxic frame by a Kopf

carrier, positioned as described by Lopez et al. (1999), and

then advanced using a microdrive (Trent-Wells). After that,

we followed the procedure described by Sinex et al. (2001)

to isolate and identify single units. Single units were

identified by the constant shape and magnitude of the

spikes, which were monitored throughout the experiment.

Up to seven electrode passes with slight variations in the

point of entry were needed to encounter single units in the

cochlear root nucleus. As a control, auditory brainstem

evoked potentials were recorded prior to and after each

experiment to check the status of nerve conduction in the

auditory nerve during the experiment (Supplemental

Fig. 2). Changes in wave amplitude of auditory brainstem

evoked potentials were taken into account when analyzing

the data to assess possible auditory nerve damage due to

multiple electrodes passes.

Stimulus presentation paradigms and data collection

were controlled by a PC-compatible computer and custom

software written for MATLAB (MathWorks, Natick MA,

USA). Stimulus waveforms were digitally synthesized

online. Search stimuli were tone bursts, and records were

obtained only from well-isolated single units. At the

beginning of the experiment, characteristic frequency and

threshold were measured from the frequency tuning curves

which were acquired with an automated procedure that

estimated threshold at many closely spaced frequencies.

Frequency response maps (combination of frequencies and

intensities capable of evoking a response) were obtained

automatically using a randomized paradigm that presented

a set of frequency/intensity pairings specifically chosen to

define the complete details of a unit’s frequency selectivity.

The response map consisted of a set of spike counts

obtained at 6 sound pressure levels (SPLs) and 63 fre-

quencies. After the collection of tuning curve and response

maps, pure tones were used as experimental stimuli to

perform more detailed measurements of unit responses to

assess the modifications by prepulse presentation at dif-

ferent conditions (duration, intensity and interstimulus

intervals).

In the probes designed to test the auditory prepulse

inhibition, two consecutive pure tones of the characteristic

frequency (CF) for the units investigated were presented

ipsilaterally to the recording site. The spikes times evoked

by the different stimuli were stored and were used to cal-

culate the response magnitude (spikes per trial) and first-

spike latencies. In each probe, we obtained peristimulus

time (PST) histograms and dot rasters to display the

responses to 50–100 repetitions of the tones. Intensity,

duration and interstimulus intervals (ISI) of the pulse and

prepulse tones were shown in each PST histogram. The bin

size for the PST histograms was 1.4 ms, and was reduced

to 0.3 ms in the expanded views.

Results

VNTB efferent pathways toward the cochlear root

nucleus

To determine the course and termination of descending

axons from the ventral nucleus of the trapezoid body

(VNTB) to the cochlear root nucleus, we injected BDA

into the rostral end of the VNTB. BDA injections into the

VNTB were small (0.3–0.6 mm in diameter), round in

shape, and were located in the rostral end of the nucleus

without spreading to adjacent superior olivary nuclei

(Fig. 1a). Following BDA injections in the VNTB, we

found two axonal pathways projecting toward the cochlear

root nucleus (Fig. 1). The first pathway consisted of thin

fibers coursing bilaterally through the trapezoid body

(TB). These BDA-labeled fibers have a diameter of

approximately 2 lm and projected to the cochlear root

nucleus of both sides. VNTB axons in the TB were clearly

distinguishable from thicker axons (6–8 lm in diameter)

of CRNs which were immunostained for CaBP (Fig. 1b).

The second track of VNTB axons was identified as the

medial olivocochlear bundle (OCB). We observed that

smooth-labeled fibers (*1.3 lm in diameter) projected

bilaterally from the injection site in the left VNTB. The

contralateral labeled fibers cross beneath the floor of the

fourth ventricle (Fig. 1c), running through the cochlear

nerve to project predominantly to the contralateral cochlea

and terminate on the outer hair cells (Fig. 1d). As

described in our previous study (Gomez-Nieto et al.

2008a), we found that VNTB neurons massively innervate

CRNs of both sides (Fig. 1e, f). To visualize the mor-

phology of the CRNs including cell bodies, dendrites and

axons, we immunostained brainstem sections for CaBP.

CRNs showed strong positive immunolabeling for CaBP

(Fig. 1e, f) as previously described by Lopez et al. (1993).

BDA-labeled axons from the VNTB enter along the

cochlear root nucleus, where they terminate in numerous

endings onto the cell bodies and dendrites of CRNs in a

bead-like pattern (Fig. 1e). We can infer from these

results that two possible populations of VNTB neurons

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might be the source of direct inputs on the CRNs: (1)

Medial olivocochlear (MOC) neurons which project

bilaterally to the outer hair cells in the cochlea through

the OCB, and (2) Non-olivocochlear neurons which

project bilaterally through the trapezoid body (TB). We

performed a comparative analysis of BDA track-tracing

experiments injected at multiple sites to elucidate which

VNTB neuronal type innervates the CRNs.

Fig. 1 Neuronal efferent pathways from the ventral nucleus of the

trapezoid body (VNTB) to the region of the cochlear root neurons

(CRNs). a Micrograph of a Nissl-stained coronal section shows a

representative case with injection site (IS) in the rostral part of the

VNTB. A higher magnification of the IS (corresponding to the frame)

is shown below. b Micrograph of the trapezoid body (TB) shows thin

anterogradely labeled axons (arrowheads) after the BDA injection

shown in Fig. 1a. VNTB-labeled axons (in black) were distinguish-

able from thick CRNs axons (arrows) labeled by CaBP immunohis-

tochemistry (in brown). c Medial olivocochlear axons labeled with

BDA crossing beneath the floor of the fourth ventricle (4 V). The

inset shows a higher magnification of a crossing medial olivocochlear

axon (arrowhead). d Micrograph shows medial olivocochlear axons

and endings in the contralateral cochlea after BDA injection in the

VNTB. The inset shows a higher magnification of the BDA-labeled

endings on the outer hair cells (OHC, arrow). The lack of labeled

terminals on the inner hair cells (IHC) indicates that the IS did not

spread to the lateral superior olive. e Micrograph shows the cochlear

root nucleus after CaBP immunostaining. f Montage of 6 micrographs

(corresponding to the boxed area in E) shows details of VNTB

terminals on cell bodies (arrowheads) and dendrites (arrows) of a

CRN immunopositive for CaBP. Note the characteristic bead-like

pattern of VNTB axons in the cochlear root nucleus. MNTB medial

nucleus of the trapezoid body, py pyramidal tract, RtTg reticulo

tegmental nucleus, Sp5 spinal trigeminal nucleus, VCN ventral

cochlear nucleus. Scale bars 2 mm in a (1 mm in the image below);

50 lm in b; 200 lm in c (20 lm in the inset); 100 lm in d (25 lm in

the inset); 500 lm in e; 20 lm in f

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VNTB neurons projecting to CRNs do not send axon

collaterals into the VCN

To determine whether MOC neurons innervate the CRNs,

two animals received BDA injections into the medial OCB

at two different locations. In the first animal, the injection

had a small, elongated shape (0.2–0.3 mm in diameter) and

was located deep in the cochlear nerve just where medial

OCB fibers run into the periphery toward the cochlea

(Fig. 2a). BDA-labeled fibers were located in a bundle

within the vestibular root and axon collaterals entered the

ventral cochlear nucleus (VCN, Fig. 2b). OCB fibers

labeled with BDA were also observed crossing the mid-line

at the floor of the fourth ventricle (Fig. 2c). The tracer

uptake by these axons resulted in retrograde labeling of

MOC neurons (e. g. in Fig. 2d). The non-olivocochlear

efferent pathway of the VNTB was not filled with tracer as

we did not observe any labeled structure in the TB (Fig. 2a,

d, e). In the second animal, a small injection (0.3 mm in

diameter) was made, without spreading to the adjacent

VCN, in the course of OCB axons just before they enter the

cochlear nerve root. As in the first case, we found the

typical distribution of medial OCB fibers (see below

Fig. 5a–c). In both cases with BDA injections into the

OCB, there was an absence of labeled boutons on the

CRNs (Fig. 2e–g). Thus, there is a strong likelihood that

the MOC neurons do not innervate the CRNs.

In addition to the MOC projection, VNTB non-olivo-

cochlear neurons project to almost the entire rostrocaudal

extent of the VCN by way of the trapezoid body of both

sides (Warr and Beck 1996). To determine if the beaded

endings on the CRNs from the VNTB (Fig. 1e) arise as

terminal branches from axons of these neurons, we injected

BDA into the left VCN. The BDA injection site was round

(0.4 mm in diameter) and included both posteroventral and

anteroventral areas of the VCN (Fig. 3a). Due to the

bidirectional nature of the transport of the BDA, we found

anterograde labeling in the form of a thin band of axons

and swellings in the ipsilateral lateral superior olive as well

as retrogradely labeled neurons in the VNTB (Fig. 3b;

Supplemental Fig. 3). Neurons that were retrogradely

labeled in the VNTB varied in size and dendritic pattern

(Fig. 3b, d) suggesting that both medial olivocochlear and

non-olivocochlear neurons send axonal terminals into the

VCN. Accordingly, we observed labeled axons in the TB

(Supplemental Fig. 3) and in the medial OCB (Fig. 3c). In

the ipsilateral cochlear root nucleus, our material showed

large synaptic terminals (4–7 lm in diameter) that pre-

sumably correspond to collaterals of auditory primary

afferents which followed a straight course toward the

center of the VCN (Fig. 3e, f). These large terminals were

entirely different in size and distribution to those present in

the cochlear root nucleus after BDA injection in the

VNTB. We did not find the typical en passant and bead-

like pattern of VNTB axons in the cochlear root nucleus

(Fig. 3e, f; Supplemental Fig. 3), suggesting that VNTB

inputs onto the CRNs do not arise from VNTB neurons that

also target the VCN.

Non-olivocochlear neurons send direct projections

to CRNs via the trapezoid body

The results described above suggest that a subpopulation of

non-olivocochlear cells are the source of VNTB inputs on

the CRNs. To verify the pathway that VNTB axons follow

before entering the cochlear root nucleus, we injected BDA

into the TB. The injection sites varied in size (0.4–1 mm in

diameter) and were confined to the TB in an area which

was beneath and in close proximity to the superior olivary

complex (Fig. 4a, b). As expected, we observed BDA-

labeled axons in the TB (Fig. 4c; Supplemental Fig. 4).

Many of these BDA-labeled fibers were thick and resem-

bled those axons of CRNs immunostained for CaBP,

whereas other axons were thinner with the same diameter

as those observed in the TB after BDA injections in the

VNTB. We followed the directions of both types of axons

and found that they turn ventrally to enter the cochlear root

nucleus. The thicker axons emerged from retrogradely

labeled CRNs somata (Fig. 4d, e; Supplemental Fig. 4),

and the thinner fibers terminate as numerous endings onto

the cell bodies and dendrites of CRNs in a bead-like pattern

(Fig. 4d, f; Supplemental Fig. 4). Thus, VNTB axons

innervating the CRNs followed the same course as CRNs

axons, running through the TB. As in cases with BDA

injections in the VNTB, the labeled en passant boutons

distributed along the entire course of the fibers from the

dorsal to the most ventral divisions of the cochlear root

nucleus (Supplemental Fig. 4). In this set of experiments,

we did not encounter any BDA-labeled structures in the

medial OCB or terminals on the outer hair cells of the

cochlea (Fig. 4g).

To fully confirm the source of VNTB inputs to CRNs, a

mechanical lesion of the OCB was made in the rostro-

caudal plane after injecting BDA in the left VNTB. To

assess the effectiveness of the lesion, the cochleae were

analyzed histologically to detect any BDA labeling. Fig-

ure 5 illustrates an animal with BDA injection in the OCB

(Fig. 5a–c) and a representative case with OCB transection

after BDA injection in the VNTB (Fig. 5d–h). In the

lesioned cases, the cut was placed off-midline and con-

tralaterally to the injection site (Fig. 5d). As compared to

the animal with OCB labeling, the lesioned cases presented

a lack of BDA-labeled axons in the crossed OCB (Fig. 5b,

e). We did not find boutons on CRNs after OCB labeling,

however, the lesioned animals showed VNTB-labeled ax-

ons that give off boutons on CRNs of both sides (Fig. 5c,

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f). These BDA-labeled endings distributed in a bead-like

pattern onto the somata and dendrites of CRNs (Fig. 5g, h),

suggesting that VNTB inputs to CRNs originate exclu-

sively from non-olivocochlear neurons. A detailed sum-

mary of the neuroanatomical results is provided in Table 1

which contains the distribution and density of the fibers and

boutons observed in each set of experiments. Comparing

all neuroanatomical findings, we conclude that a distinct

subset of non-olivocochlear neurons send bilateral projec-

tions via the trapezoid body to directly innervate the CRNs.

Gene expression of muscarinic and nicotinic receptors

in the cochlear root nucleus

The cholinergic system plays an important role in the

inhibition of the acoustic startle reflex (Fendt et al. 2001)

through activation of muscarinic and nicotinic receptors

(Jones and Shannon 2000; Schreiber et al. 2002; Bosch and

Schmid 2008). Since VNTB neurons projecting to the

CRNs use acetylcholine as their principal neurotransmitter

(Gomez-Nieto et al. 2008a), we focused on determining the

Fig. 2 Medial olivocochlear neurons do not innervate cochlear root

neurons (CRNs). a Micrograph of a coronal section shows a BDA

injection site (IS) in the ventral part of the cochlear nerve root. The

lack of axonal labeling in the trapezoid body (TB) indicates no tracer

uptake by axons of non-olivocochlear neurons. b BDA-labeled fibers

in the bundle within the vestibular root (asterisk) and labeled axon

collaterals in the ventral cochlear nucleus (VCN). The inset shows a

higher magnification of labeled terminals arising from the axon

collateral (arrowhead). c Medial olivocochlear axons labeled with

BDA crossing beneath the floor of the fourth ventricle (4 V). The

inset shows a higher magnification of a crossed medial olivocochlear

axon (arrowhead). d Micrograph of a retrogradely labeled neuron in

the ventral nucleus of the trapezoid body (VNTB). The somatic size

(144. 2 lm2 with major and minor axes of 17, 4 and 10, 6 lm) and

pattern of dendrites fit into the category of medial olivocochlear

neurons. Notice the absence of axonal labeling in the TB. e Low-

magnification micrograph shows a Nissl-stained section containing

the cochlear root nucleus. f, g Higher magnification of the dorsal

region (f) and ventral region (g) of the cochlear root nucleus

(corresponding to the frames in e). Notice the absence of labeled

terminals on the CRNs. Sp5, spinal trigeminal nucleus. Scalebars 500 lm in a; 100 lm in b, d; 200 lm in c, e; 20 lm in b,

c insets; 50 lm in f, g

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gene expression of cholinergic receptors in the cochlear

root nerve. We performed RT-PCR analysis for 9 selective

genes (Supplemental Fig. 1) and found that the cochlear

root nerve expressed 5 members of muscarinic acetylcho-

line receptors (M1-5; Fig. 6) as reported in our previous

study (Gomez-Nieto et al. 2008b). Furthermore, we also

Fig. 3 VNTB neurons innervating the ventral cochlear nucleus

(VCN) do not send axon collaterals to cochlear root neurons (CRNs).

a Micrograph of a coronal section shows a BDA injection site (IS) in

the VCN. Notice labeled axons in the trapezoid body (TB). b Nissl-

stained section of the superior olivary complex shows anterograde

labeling in the ipsilateral lateral superior olive (LSO) and retrogradely

labeled neurons in the ventral nucleus of the trapezoid body (VNTB).

The upper inset in b illustrates a higher magnification of the

characteristic projection pattern of VCN cells in the LSO (arrow-head). The inset below shows a detail of a VNTB neuron retrogradely

labeled with BDA (arrow, somatic size: 148. 5 lm2 with major and

minor axes of 15, 6 and 12, 1 lm). c Micrograph shows crossed

medial olivocochlear axons labeled with BDA. The inset (position

denoted with an arrowhead) is a higher magnification of a medial

olivocochlear axon traveling beneath the floor of the fourth ventricle

(4 V). d Micrograph shows a medial olivocochlear neuron retro-

gradely labeled with BDA (somatic size: 178. 8 lm2 with major and

minor axes of 23, 4 and 9, 7 lm). The variety in size and dendritic

pattern of retrogradely labeled VNTB neurons shown in b and

d. e Nissl-stained section of the ipsilateral cochlear root nerve.

f Higher magnification of the cochlear root nucleus (corresponding to

the frame in e) shows large axosomatic terminals (arrowhead) on

CRNs. These labeled terminals (perimeter: *19 lm) emerged at

right angles from short axon collaterals of primary auditory axons

(asterisk). The typical bead-like pattern of VNTB axons in the

cochlear root nucleus was not obtained after the BDA injection in the

VCN. MSO medial superior olive, Sp5 spinal trigeminal nucleus.

Scale bars 250 lm in a; 200 lm in b, c, e; 50 lm in d, f; 20 lm in

b, c inset

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detected the expression of 4 members of nicotinic acetyl-

choline receptors (Nb4, Nb7, Nb2 and Nb3; Fig. 6). All

these genes have a marked expression level in cells of the

prefrontal cortex and hippocampus, and hence, they were

used as positive control. In the cochlear root nucleus, the

expression levels of muscarinic (M1, M2, M4 and M5) and

nicotinic acetylcholine receptors (Na4, Nb2 and Nb3) were

relatively high. The lowest expression levels among the

muscarinic and nicotinic acetylcholine receptors corre-

sponded to M3 and Na7 receptor subunits, respectively.

These data suggest that CRNs contain a cholinergic

receptor profile sufficient to mediate the auditory prepulse

inhibition of CRN responses via the descending projection

from the VNTB to the cochlear root nucleus.

Auditory prepulse inhibition of CRN responses

Our neuroanatomical results and gene expression analysis

indicated that the cochlear root nucleus receives direct

inputs from auditory cholinergic neurons and expresses all

Fig. 4 Bead-like terminals on the cochlear root neurons (CRNs) arise

from axons that course through the trapezoid body (TB). a Schematic

drawing of a BDA injection site (IS) into the region of the TB. The

gray area displays the region of BDA diffusion. b Micrograph of a

Nissl-stained section shows the injection site in the TB. c Low-

magnification micrograph shows the cochlear root nucleus after BDA

injection in the TB and CaBP immunostaining. Note BDA-labeled

axons of different sizes coursing through the TB. d Higher magni-

fication of the cochlear root nucleus (corresponding to the frame in c)

shows BDA-retrograde and anterograde labeling after injection in the

TB. e, f Details of a BDA retrogradely labeled CRN somata and

BDA-labeled terminals on CRNs immunopositive for CaBP (position

denoted with an asterisk and arrowheads in d, respectively).

g Micrograph shows an absence of label in the cochlea after BDA

injection in the TB. IHC inner hair cells, LSO lateral superior olive,

MNTB medial nucleus of the trapezoid body, MSO medial superior

olive, OHC outer hair cells, py pyramidal tract, Sp5 spinal trigeminal

nucleus, VCN ventral cochlear nucleus, VNTB ventral nucleus of the

trapezoid body, 7n, facial nerve. Scale bars 2 mm in a; 1 mm in b,

200 lm in c; 50 lm in d; 20 lm in e and f; 100 lm in g

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necessary muscarinic and nicotinic receptors subunits to

mediate the inhibition of CRN response by auditory pre-

pulses. To determine whether CRN responses are reduced

by auditory prepulse stimulation, we delivered weak

auditory stimuli (prepulses) presented before strong audi-

tory stimuli (pulses) that evoked a full CRN response.

Portions of this electrophysiological study were presented

in preliminary form at the 15th International Symposium

on Hearing (Gomez-Nieto et al. 2010). A total of 18

auditory single units were recorded in four animals as the

electrode was advanced downward along the VCN and into

the cochlear root nucleus. Of these, 3 units were identified

as CRNs based on the electrophysiological criteria estab-

lished by Sinex et al. (2001). The other 15 units were

recorded when the electrode passed from dorsal to ventral

through the VCN and were classified as one of the major

Fig. 5 Non-olivocochlear

neurons send direct projections

to cochlear root neurons (CRNs)

via the trapezoid body (TB).

a Micrograph of a Nissl-stained

coronal section shows the

medial olivocochlear bundle

(OCB) labeled with BDA. The

injection site is shown

schematically in the inset.

b Thick and smooth BDA-

labeled fibers (arrows) coursing

bilaterally through the medial

OCB. c Micrographs show

medial OCB fibers labeled with

BDA (arrows) in the ipsilateral

and contralateral cochlear root

nucleus. Notice fibers do not

give off endings onto the CRNs

(asterisks). d Nissl-stained

section shows a mechanical

lesion (arrows) in the medial

OCB after BDA injection into

the VNTB (inset). e Micrograph

shows the lack of BDA-labeled

fibers in the OCB after the

lesion. f Low-magnification

micrograph shows the ipsilateral

and contralateral cochlear root

nucleus immunostained for

CaBP. g, h Higher

magnification of the ipsilateral

(g) and contralateral (h) region

of the cochlear root nucleus

corresponding to the boxed area

in f. Note beaded axons labeled

with BDA giving off numerous

endings (arrowheads) onto

CRNs labeled by CaBP

immunohistochemistry. VCNventral cochlear nucleus, VNTBventral nucleus of the trapezoid

body. Scale bars 1 mm in

a and d; 500 lm in b and e;

200 lm in c; 100 lm in f;20 lm in g and h

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cell types of the VCN (primary-like, chopper, pauser, and

on-sustained neurons) according to their temporal

response patterns in peristimulus time (PST) histograms.

CRNs exhibited a secure response with first-spike laten-

cies of approximately 2.2 ms (Fig. 7a, b). Figure 7b shows

a representative example of spike waveforms from an

isolated single unit. The characteristic frequencies of the

recorded CRNs were about 30 kHz, and the tuning curves

were sharp with a threshold of 10 dB sound pressure level

(SPL) (Fig. 7c, e, f). In the same animal, we found that

first-spike latencies and characteristic frequencies of

CRNs deviated markedly from units in the overlying

portions of the VCN (Fig. 7c–f). After electrophysiologi-

cal identification of the isolated units, we tested the effects

of auditory prepulses. The frequency of the prepulse and

the main pulse always matched the characteristic fre-

quency of the recorded unit, and the time between both

stimuli (interstimulus intervals: ISIs) was varied. In all

cases, the responses of CRNs were strongly inhibited by

auditory prepulse stimuli (Fig. 8). We systematically

observed a manifest decrease in the first-spike discharge

which yielded a reduction in the CRN overall response.

For example, the response of the CRN shown in Fig. 8a

was drastically reduced by prepulses which were presented

at 150 ms of ISI and with an intensity of just 15 dB SPL

over the threshold. Although this unit responded weakly

with a few spike discharges to the onset of the prepulse,

the response to the pulse was decreased by 50 %. This

result highlighted the very strong influence of the prepulse

on the CRN’s response. In a different probe, we tested the

effect of the prepulse intensity by increasing the level of

the prepulse in steps of 20 dB up to a maximum of the

pulse intensity (Fig. 8b). We kept prepulse duration

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Fig. 6 Different gene expression levels of muscarinic acetylcholine

receptors (M1-5) and nicotinic acetylcholine receptors (Na4, Na7,Nb2 and Nb3) in the cochlear root nerve (CRN), prefrontal cortex

(PFC), hippocampus (H) and negative control (C-) in the exponential

phase of different PCRs (30 cycles). Primer sequences, sizes of the

distinct fragments and conditions of PCRs are detailed in supple-

mental figure 1. b-actin was used as control for equal amounts in RT-

PCR reactions

Brain Struct Funct

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(50 ms) and interstimulus interval (100 ms) fixed during

this probe. As the prepulse intensity was increased, we

observed a reduction of the CRN response to the onset of

the pulse (Fig. 8b). The spike discharges to the pulse were

maximally inhibited (nearly 90 %) with a prepulse deliv-

ered at the same intensity as the pulse (45 dB SPL). In the

probe to test the effect of the prepulse duration, we

increased the durations of the prepulse while an intensity of

45 dB SPL and an ISI of 150 ms were kept fixed (Fig. 8c).

The loss of spikes to the onset of the pulse was produced by

increasing durations of the prepulse. The strongest inhibi-

tory effects (80 %) were observed with a prepulse duration

equal to the pulse duration (50 ms). We also examined the

effect of the ISI by decreasing the time between the pre-

pulse and the pulse (Fig. 8d). In this probe, the pulse that

evoked a full CRN response was preceded by a low-

intensity prepulse (25 dB SPL). It is shown in PST histo-

grams of Fig. 8d that CRN responses were significantly

reduced as the ISI decreased, and spikes to the onset of the

pulse were totally abolished when the prepulse was deliv-

ered just before the pulse (50 ms of ISI). Thus, the

responses of CRNs were dependent on the prepulse

intensity, the prepulse duration and the interstimulus

intervals.

CRNs differ from VCN neurons in their response

to auditory prepulse stimulation

Our neuroanatomical experiments showed that non-olivo-

cochlear neurons of the VNTB project to CRNs without

sending axons collaterals into the VCN. If only this direct

projection is involved in auditory prepulse inhibition of

CRN responses, it might be expected that VCN neurons

would not have the same sensitivity. Therefore, a total of

15 single units of the VCN were subjected to the same

probes previously performed on CRNs. None of the VCN

units exhibited reduction of spike discharges comparable to

those observed in CRNs. As a reference, Fig. 9a shows a

PST histogram in which the responses of a CRN unit and a

VCN chopper unit were merged. Those units were recorded

in the same animal along the same electrode track, and

were subjected to auditory prepulse stimulation using

identical prepulse and pulse parameters (intensity, dura-

tion, and ISI) presented at the respective neuron’s charac-

teristic frequency. The VCN chopper unit maintained the

number of spikes per trial and the PST histogram-shape as

the prepulse intensity increased. On the contrary, the

response of the CRN unit was drastically reduced by

auditory prepulse stimulation (Fig. 9a). For comparison,

we further recorded the response of CRN and VCN units by

presenting auditory prepulse stimuli at many prepulse

intensities and ISIs (Figs. 9b–f). The number of spikes of

CRN units to the onset of the pulse was reduced as prepulse

intensity increased and ISIs decreased. This inhibitory

effect was maximal when prepulses were presented at high-

intensity level and short ISI (Fig. 9b). Conversely, VCN

units that received auditory prepulse stimulation did not

show such loss in the ratio of the number of spikes. Various

classes of VCN neurons including chopper, primary-like,

pauser, and long-latency neurons showed no evidence of

the inhibition observed in CRNs (Fig. 9c–f). This suggests

that CRNs, unlike any other type of neuron in the VCN, are

evolved to participate in auditory prepulse inhibition.

Discussion

In the present study, we provide insights into the structure

and function of the cholinergic VNTB–CRNs projection

which was described in our previous report (Gomez-Nieto

et al. 2008a). We determined the type of VNTB neuron

which innervates the CRNs and the pathway followed by

the axons to the cochlear root nucleus. Thus, we showed

that non-olivocochlear neurons from the VNTB send direct

and bilateral protections to the cochlear root nucleus via

the trapezoid body. From this result, we can infer that

CRNs receive cholinergic auditory inputs from a system

which is not involved in the descending control of the

cochlea. This led us to question the functional role of

cholinergic efferent innervation in the cochlear root

nucleus, an essential element of the primary acoustic startle

pathway (Lopez et al. 1999). Our results also showed that

CRNs possess muscarinic and nicotinic receptors which are

involved in the inhibition of startle mediating PnC neurons

(Jones and Shannon 2000; Fendt et al. 2001; Bosch and

Schmid 2006). Finally, our electrophysiological study

showed that the neuronal activity of CRNs is strongly

inhibited by auditory prepulses, suggesting the existence of

at least two cholinergic pathways that modulate the pri-

mary acoustic startle circuit.

The VNTB neuronal type which project to the cochlear

root nucleus

The neuronal heterogeneity of the VNTB is consistent

with its multiple efferent projections to a large variety of

structures. Besides its projection to each cochlea, VNTB

has axonal terminations in the dorsal and ventral cochlear

nucleus of both sides, the contralateral lateral superior

olive and the ipsilateral IC (Warr and Beck 1996). Our

previous report demonstrated that VNTB also sends

bilateral projections to the cochlear root nucleus (Gomez-

Nieto et al. 2008a). One of the main goals of the present

study was to elucidate the VNTB neuronal type which

projects to CRNs. This was a complex issue considering

that some types of VNTB neurons project with branching

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Fig. 7 Electrophysiological identification of cochlear root neurons

(CRNs) and ventral cochlear nucleus (VCN) neurons. a Dot rasters

(top) and PST histograms (bottom) of spikes elicited by a 26 kHz,

40 dB SPL tone. Notice the typical discharge pattern ‘‘primary-like

with notch’’ of a CRN. b Expanded view (corresponding to the frame

in a) of the first 10 ms after tone onset of the response shows a first-

spike latency of 2.2 ms approximately. No correction for the acoustic

delay of approximately 0.5 ms was made. A sample of overlaid spike

waveforms is shown on the top. c, d Frequency response map of a

cochlear root unit (c) and a ventral cochlear nucleus unit (d) analyzed

in the study. Color-scale plots indicate the magnitude of response to

tones at many different frequencies and SPLs. e, f Recording

locations of 18 units analyzed in the study. Graphs show characteristic

frequency versus latency (e) and characteristic frequency versus the

depth in microns (f) where cochlear root units were found. The

symbols indicate the units obtained from different cases (asterisk,

case 0504; filled circle, case 0508; filled triangle, case 0506; filledsquare, case 0505). Locations dorsal to the target are indicated by

negative numbers. The colored circles represents the two nuclei

where the units where located. CRNs exhibit high CF (*30 kHz) and

short first-spike latency (*2 ms) compared to cochlear nucleus units.

The CRN and VCN units denoted with blue and red arrowscorrespond to the frequency response maps shown in c, d,

respectively

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axons to more than one VNTB target (Warr and Beck

1996). Since the VNTB projects to the cochlear root

nucleus bilaterally with approximately equal magnitude

(Gomez-Nieto et al. 2008a), we rejected in the first

instance VNTB neurons with homolateral projections as a

possible source of inputs to the cochlear root nucleus. To

support this assumption, we reviewed our previous studies

with BDA injection in the IC (e. g. case 05/180 in

Gomez-Nieto et al. 2008a) and we did not observe any

labeled boutons on the CRNs. This is also confirmed by

Warr and Beck (1996) who reported that VNTB neurons

projecting to IC never crossed the midline. Our compar-

ative analysis of BDA-labeled structures after injections

into the axonal projections of VNTB descending pathways

(OCB and TB) and the VCN (see details in Table 1)

indicated that a subset of non-olivocochlear neurons sends

a direct projection to CRNs via the trapezoid body. BDA

was used as neuronal tracer in all these experiments. BDA

is an effective bidirectional pathway tracer which has

excellent collateral–collateral transport within a single cell

(Rajakumar et al. 1993; Chen and Aston-Jones 1998), that

is, dextrans can fill the entire cell with injection into the

Fig. 8 Auditory prepulse inhibition of neuronal activity in the

cochlear root nucleus. The prepulse and pulse parameters (intensity,

duration and interstimulus intervals) used in the probes were showed

in the upper part of each PST histogram. a PST histograms showing

the effects of prepulse tones (gray line) on the CRN response to pulse

tones (black line). Notice the reduction of the spikes per trial (arrow)

after prepulse stimulation. b PST histograms for three prepulse

stimulation probes show the effects of the prepulse intensity level on

the discharge of a CRN. Arrows indicate the reduction of the spikes

per trial with the increase of prepulse intensity. c PST histograms for

three prepulse stimulation probes show the effects of the prepulse

duration on the discharge of a cochlear root neuron. Arrows indicate

the reduction of the spikes per trial as the prepulse duration increased.

d PST histograms for four prepulse stimulation probes show the

effects of the interstimulus interval (ISI) on the discharge of a CRN.

Arrows indicate the reduction of the spikes per trial as ISIs decreased

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terminal field of a single collateral (Doucet and Ryugo

2003; Gomez-Nieto and Rubio 2009). Thus, our com-

parative analysis provided several lines of evidence that

support non-olivocochlear neurons as the principal source

of VNTB inputs to CRNs:

1. Our BDA injections in the OCB did not generate

anterograde labeling of boutons on the CRNs. In these

cases, we obtained retrogradely labeled neurons in the

VNTB that had the morphology and size of MOC

neurons (Vetter and Mugnaini 1992; Cantos et al.

2000), and we did not observe any labeled fibers in the

trapezoid body. This indicates that our OCB injections

only filled axons of MOC neurons and that they did not

project to CRNs. Consistently, previous studies which

had traced the axonal pathway of MOC neurons did

not report any evidence of collateral branches into the

rat cochlear root nucleus (White and Warr 1983; Vetter

and Mugnaini 1992; Horvath et al. 2000; Cantos et al.

2003; Warr and Beck 1996; Warr and Boche 2003;

Gomez-Nieto et al. 2008a).

2. It is presently known that MOC neurons have collat-

erals to the VCN (Osen et al. 1984; Brown et al. 1988;

Warr and Beck 1996; Horvath et al. 2000; Brown and

Vetter 2009). If those MOC neurons also send

collaterals into the cochlear root nucleus, we might

encounter labeled terminals on the CRNs after BDA

injections into the VCN. However, our BDA injections

into the VCN gave a negative result. In these

experiments, we observed an anterograde pattern of

labeling in the lateral superior olive equal to that

described by Doucet and Ryugo (2003). This obser-

vation indicated that the tracer was taken up by VCN

cells and confirmed the effectiveness of our injection

site which was precisely located where OCB branches

terminated within the VCN (Horvath et al. 2000;

Fujino and Oertel 2001). In these cases, we assured

Fig. 9 CRNs differ from VCN

neurons in their response to

auditory prepulse stimulation.

a Merged PST histograms of a

CRN unit (displayed in black)

and a chopper unit from the

ventral cochlear nucleus

(displayed in white). The

prepulse stimulation probes are

the same for both neuron types

(except that each unit was

stimulated by a tone at its own

CF). The CRN reduced its

number of spikes during

prepulse stimulation (arrows),

but the chopper unit did not. b–

f Plot shows number of spikes

versus interstimulus intervals

(ISIs) for one CRN unit (b), and

four VCN units: a chopper unit

(c), a primary-like unit (d), a

pauser unit (e), and a long-

latency unit (f) after different

stimulation parameters (S1,

prepulse; S2, pulse) indicated in

the legend at lower left corner

of each plot. The CRN reduced

its response as prepulse

intensity increased and

interstimulus intervals

decreased. In contrast, the

responses of all other ventral

cochlear units were independent

of prepulse intensity and

interstimulus interval

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that MOC neurons were completely filled with the

tracer as our results showed labeled crossed OCB

fibers under the fourth ventricle and retrogradely

labeled neurons which fulfilled the morphological

criteria established for MOC neurons (Vetter and

Mugnaini 1992; Cantos et al. 2000). Following our

BDA injections into the VCN, we did find labeled

terminals on the somata of CRNs. These labeled

axosomatic terminals were only observed ipsilateral to

the injection site and differed from VNTB terminals in

size and axonal branching pattern (Gomez-Nieto et al.

2008a). As reported in earlier studies, their large size

and axonal branching pattern resembled that of

terminals immunolabeled for the vesicular glutamate

transporter 1 which arise from collaterals of auditory

primary axons (Osen et al. 1991; Lopez et al. 1993;

Gomez-Nieto et al. 2008b).

3. The lesion of the OCB after BDA injection in the

VNTB confirmed the non-olivocochlear origin of the

VNTB–CRNs projection. Previous studies demon-

strated that mechanical lesions of the OCB near the

floor of the fourth ventricle were effective in elimi-

nating efferent terminals of MOC neurons (Liberman

1990; Bledsoe et al. 2009). In our material, the

transection of crossed olivocochlear fibers was made

at this particular location and the cochlea was analyzed

histologically to assess the completeness of the lesion.

Our results showed VNTB bead-like axons on CRNs

after the OCB lesion suggesting that the VNTB–CRNs

projection followed a non-olivocochlear pathway. The

same conclusion arises from our injections in the TB

which indicates that VNTB axons innervate CRNs

course via the trapezoid body. This course appears to

be similar to the initial trajectory of CRN axons in the

trapezoid body (Lopez et al. 1993). We verified this,

since the injections in the TB resulted in retrograde

labeling of CRNs somata as well as anterograde

labeling of VNTB terminals in the cochlear root

nucleus.

In sum, our morphology study indicates that non-oli-

vocochlear neurons from the VNTB are a significant source

of axonal terminals in the cochlear root nucleus. These

non-olivocochlear neurons appeared not to send collaterals

to brain structures receiving projections from other VNTB

neuronal types such as the VCN or the cochlea. Therefore,

it is reasonable to conclude that VNTB neurons projecting

to CRNs belong to a distinct neuronal population which do

not participate in controlling the cochlea. This makes it

likely that the VNTB–CRNs projection is involved in the

modulation of the acoustic startle reflex rather than the

medial olivocochlear reflex.

The role of the cholinergic system within the cochlear

root nucleus

Previous neuroanatomical studies have shown strong evi-

dence of acetylcholine release in the cochlear root nucleus

(Vetter et al. 1993a; Yao and Godfrey 1999; Gomez-Nieto

et al. 2008a). In the superior olivary complex, the VNTB is

the largest source of descending cholinergic inputs (Sher-

riff and Henderson 1994; Yao and Godfrey 1998). Our

group has recently demonstrated that CRNs receive their

cholinergic input from VNTB neurons (Gomez-Nieto et al.

2008a). The fact that our RT-PCR analysis showed a sig-

nificant gene expression of muscarinic and nicotinic ace-

tylcholine receptors in the cochlear root nucleus also

supported the release of acetylcholine on CRNs. These

cholinergic receptors are likely to be specific for projec-

tions from the VNTB given that injections of neuronal

tracers in the cochlear root nucleus do not generate retro-

grade labeling in any other cholinergic nuclei (Gomez-

Nieto et al. 2008a). It is presently known that acetylcholine

plays a crucial role in mediating PPI of the acoustic startle

response (reviewed in Fendt et al. 2001). Several studies

have shown that muscarinic receptors, particularly the

subtypes M2 and M4, are involved in PPI mediation at the

level of PnC (Koch et al. 1993; Fendt and Koch 1999;

Jones and Shannon 2000; Bosch and Schmid 2006). Thus,

activation of M2 and M4 receptors subtypes mediates a

strong inhibition of PnC giant neurons (Bosch and Schmid

2006, 2008). Our data showed that CRNs also expressed

M2 and M4 subtypes as confirmed by immunolabeling in

Gomez-Nieto et al. (2008b), and hence, acetylcholine

might have also an inhibitory effect on CRNs. Although the

role of nicotinic receptors in PPI is less studied, several

pharmacological experiments have shown, with contro-

versial results, that stimulation of nicotinic receptors

affects sensorimotor gating (Acri et al. 1994; Schreiber

et al. 2002). In fact, the investigation of the effects of

different nicotine receptors agonist and antagonist on PPI

supports the involvement of the nicotinic receptor subtypes

a4 and b2, and possibly a7 in the control of PPI (Schreiber

et al. 2002; Suemaru et al. 2004). Our result showed that

CRNs contain both a and b subunits, and more specifically,

the a4 and b2 subtypes which were expressed in a rela-

tively high level. Some evidence also suggests that these

nicotinic receptors play a role in a faster PPI pathway

acting at short interstimulus intervals (Bosch and Schmid

2008). This idea together with the fact that CRNs exhibit

all the molecular cholinergic machinery involved in PPI

mediation conforms well with our initial hypothesis: the

VNTB projection might inhibit startle signaling in the

cochlear root nucleus through a cholinergic mechanism.

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The observed inhibition of CRN responses after auditory

prepulse stimulation, particularly strong when short inter-

stimulus intervals were used, is also consistent with that

suggestion.

Multiple pathways for the auditory inhibition

of the primary acoustic startle circuit

One of the most accepted anatomical models for explaining

the circuitry mediating auditory PPI includes three serially

connected midbrain structures: the IC, the superior col-

liculus, and the pedunculopontine tegmental nucleus which

sends inhibitory cholinergic inputs to the PnC (reviewed in

Fendt et al. 2001). Yet, it is important to note that not all

properties of the PPI such as the shorter ISIs in which this

paradigm occurs (20 ms, Hoffman and Ison 1980) are

explained by the proposed circuit. Recent studies support

the existence of additional pathways that bypass some

components of this long multimodal circuit to mediate fast

auditory PPI (Yeomans et al. 2006; Gomez-Nieto et al.

2008a; reviewed in Li et al. 2009). The cochlear root

nucleus is an obligatory relay station in the primary

acoustic startle pathway (Lee et al. 1996; Lopez et al.

1999), and its neurons possess a tremendous similarity with

giant neurons of the reticular formation. Both CRNs and

giant PnC neurons appear to be similar in their electro-

physiological properties (Lingenhohl and Friauf 1992;

Sinex et al. 2001), direct cholinergic afferent inputs (Fendt

et al. 2001; Gomez-Nieto et al. 2008a), and neurotrans-

mitter machinery (Bosch and Schmid 2006; Gomez-Nieto

et al. 2008b). The resemblance supports the idea that the

acoustic startle reflex might be inhibited in both nuclei of

the primary acoustic startle circuit. Our electrophysiologi-

cal results showed that CRNs responses were strongly

inhibited by auditory prepulse stimulation, particularly at

short ISIs. The reduction in the discharge pattern of CRN

responses was observed after presentation of two consec-

utive pure tones, a prepulse followed by the testing pulse,

separated by an ISI. It could be argued that the observed

inhibition was due to forward masking or the medial oli-

vocochlear reflex which might reduce cochlear gain, and

therefore, the response of primary auditory neurons.

However, our results showed that CRN responses were

drastically reduced at ISIs long enough to avoid forward

masking. Physiological studies concerning forward mask-

ing in auditory nerve and VCN units have shown that there

is no significant inhibition at ISIs longer than 100 ms

(Harris and Dallos 1979; Shore 1995). In our results, we

showed auditory prepulse inhibition of CRNs’ response at

ISIs that were varied up to 200 ms. Furthermore, if forward

masking might contribute to the response reduction at the

shortest ISIs, we should have found a similar reduction in

VCN neurons which also receive primary auditory

afferents. Our results showed that VCN units exhibited

minimal reduction of spike discharges, indicating that the

drastic reduction observed in CRNs is likely to have a more

complex basis. The number of CRN units was small

compared with the VCN units that were recorded in each

animal. This is undoubtedly a consequence of the small

size of the cochlear root nucleus, no more than 40–50

neurons (Merchan et al. 1988), and its relative inaccessi-

bility inside the internal auditory meatus. It is important to

notice that such strong inhibition in the CRNs response was

obtained after stimulations with prepulses of weak inten-

sities and short durations. This suggests that the prepulse

signals were processed by a mechanism that enhances the

effect of the prepulses on the CRNs’ response. A musca-

rinic and nicotinic cholinergic mechanism activated by the

descending cholinergic projection from the VNTB to the

cochlear root nucleus could provide that kind of inhibition.

The VNTB contains at least two distinct groups of cho-

linergic cells with descending pathways, MOC neurons and

non-olivocochlear neurons, which provide cholinergic

inputs to the cochlea and the VCN (Vetter et al. 1991;

Sherriff and Henderson 1994; Warr and Boche 2003). Our

tracer experiments indicate that a subpopulation of non-

olivocochlear neurons project solely to the cochlear root

nucleus. The fact that non-olivocochlear neurons innervate

CRNs without sending collaterals to VCN is consistent

with the electrophysiological data showing that CRNs

differ from VCN neurons in their response to auditory

prepulse stimulation. In sum, our study supports the exis-

tence of multiple neuronal pathways for mediating PPI, and

suggests that the cochlear root nucleus might be involved

in the primary circuit of the auditory PPI. In agreement

with the current models (reviewed in Fendt et al. 2001; Li

et al. 2009) and accounting for the above results, we pro-

pose an alternative neuronal pathway for the mediation of

the auditory PPI (Fig. 10). In this proposed circuit, the

CRNs might inhibit the acoustic startle signaling when the

prepulse is presented at short ISIs. Such fast inhibition

might be mediated by the VNTB–CRNs cholinergic pro-

jection which activates a muscarinic and nicotinic inhibi-

tion mechanism in the cochlear root nucleus. Similarly,

PnC neurons might receive auditory prepulse information

at long ISIs through a slow multi-modal circuit which

involves the IC, the superior colliculus, and finally the

cholinergic projection from the pedunculopontine teg-

mental nucleus to PnC (Fendt et al. 2001, Li et al. 2009).

Both pathways might be interconnected by the reciprocal

projections between the IC and the VNTB (Beyerl 1978;

Faye-Lund 1986; Coleman and Clerici 1987; Vetter et al.

1993b; Warr and Beck 1996; Gomez-Nieto et al. 2008a).

The IC–VNTB–CRNs auditory pathway and other con-

verging inputs to the CRNs like those from the locus

coeruleus, a noradrenergic attention system, might be

Brain Struct Funct

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important to understand the binaural processing observed

in both baseline PPI and attentional modulation of PPI

(Gomez-Nieto et al. 2008a, Gomez-Nieto et al. 2008b).

Acknowledgments We gratefully acknowledge Dr. Sebastian

Hormigo for critically reading an early version of the manuscript. We

thank the reviewers for their valuable comments which helped to

considerably improve the quality of the manuscript. This research was

supported by grants from the Spanish Ministry of Science and Inno-

vation (MICINN, #BFU2010-17754) to Dr. Dolores E. Lopez; the

National Institute on Deafness and Other Communication Disorders

(NIDCD, #DC00341) to Dr. Donal E. Sinex; Fundacion ‘‘Samuel

Solorzano Barruso’’ (FS/6-2012) to Dr. Ricardo Gomez-Nieto; and

the Sao Paulo State Research Foundation (FAPESP, #2008/02771-6)

to Dr. Jose de Anchieta C. Horta-Junior.

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