ORIGINAL ARTICLE

Projections from the lateral vestibular nucleus to the spinal cordin the mouse

Huazheng Liang • Timea Bacskai • Charles Watson •

George Paxinos

Received: 19 December 2012 / Accepted: 27 February 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract The present study investigated the projections

from the lateral vestibular nucleus (LVe) to the spinal cord

using retrograde and anterograde tracers. Retrogradely

labeled neurons were found after fluoro-gold injections into

both the cervical and lumbar cord, with a smaller number

of labeled neurons seen after lumbar cord injections.

Labeled neurons in the LVe were found in clusters at

caudal levels of the nucleus, and a small gap separated

these clusters from labeled neurons in the spinal vestibular

nucleus (SpVe). In the anterograde study, BDA-labeled

fiber tracts were found in both the ventral and ventrolateral

funiculi on the ipsilateral side. These fibers terminated in

laminae 6–9. Some fibers were continuous with boutons in

contact with motor neurons in both the medial and lateral

motor neuron columns. In the lumbar and sacral segments,

some collaterals from the ipsilateral vestibulospinal tracts

were found on the contralateral side, and these fibers

mainly terminated in laminae 6–8. The present study

reveals for the first time the fiber terminations of the lateral

vestibular nucleus in the mouse spinal cord and therefore

enhances future functional studies of the vestibulospinal

system.

Keywords Hindbrain � Vestibulospinal tract �Spinal cord � Motor neurons � Vestibular system

Abbreviations

2SpL Lamina 2 of the spinal gray, lateral part

3Sp Lamina 3 of the spinal gray

4Sp Lamina 4 of the spinal gray

4V 4th ventricle

5SpM Lamina 5 of the spinal gray, medial part

5SpL Lamina 5 of the spinal gray, lateral part

6Sp Lamina 5 of the spinal gray

7n Facial nerve

7N Facial nucleus

7Sp Lamina 7 of the spinal gray

8Sp Lamina 8 of the spinal gray

10Sp Area 10 of the spinal gray

Ax9 Axial muscle motoneurons of lamina 9

Bi9 Biceps motoneurons of lamina 9

C4 4th cervical segment

C5 5th cervical segment

C6 6th cervical segment

CC Central canal

contra Contralateral

cu Cuneate fasciculus

DAB 3,30-Diaminobenzidine

DC Dorsal cochlear nucleus

De9 Deltoid muscle motoneurons of lamina 9

dcs Dorsal corticospinal tract

dr Dorsal root

Fl Flocculus

GAD Glutamic acid decarboxylase

Gi Gigantocellular reticular nucleus

icp Inferior cerebellar peduncle

IMM Intermediomedial column

IntA Interposed cerebellar nucleus, anterior part

H. Liang and T. Bacskai made an equal contribution to this work.

H. Liang � T. Bacskai � C. Watson � G. Paxinos (&)

Neuroscience Research Australia, Cnr Barker Street

and Hospital Road, Randwick, NSW 2031, Australia

e-mail: g.paxinos@neura.edu.au

C. Watson

Faculty of Health Sciences, Curtin University,

Perth, WA 6845, Australia

G. Paxinos

School of Medical Sciences, The University of New South

Wales, Sydney, NSW 2052, Australia

123

Brain Struct Funct

DOI 10.1007/s00429-013-0536-4

IO Inferior olive

ipsi Ipsilateral

L3 3rd lumbar segment

L4 4th lumbar segment

L6 6th lumbar segment

Lat Lateral cerebellar nucleus

lf Lateral funiculus

LSp Lateral spinal nucleus

LVe Lateral vestibular nucleus

Med Medial cerebellar nucleus

mlf Medial longitudinal fasciculus

MVeMC Medial vestibular nucleus, magnocellular part

MVePC Medial vestibular nucleus, parvicellular part

PFl Paraflocculus

Pr Prepositus nucleus

py Pyramidal tract

rs Rubrospinal tract

scp Superior cerebellar peduncle

SI9 Supraspinatus and infraspinatus motoneurons

of lamina 9

sp5 Spinal trigeminal tract

Sr9 Serratus anterior motoneurons in lamina 9

SuVe Superior vestibular nucleus

T7 7th thoracic segment

VCPO Ventral cochlear nucleus, posterior part, octopus

cell area

vGluT2 Vesicular glutamate transporter 2

vmf Ventromedial fissure

vr Ventral root

vwc Ventral white commissure

Introduction

It has long been known that the lateral vestibular nucleus

(LVe) is involved in locomotion and postural control

through its connections with the spinal cord (Zemlan et al.

1979; Hayes and Rustioni 1981; Leong et al. 1984; Rose

et al. 1992; Masson et al. 1991; Wada et al. 1993; Auclair

et al. 1993; Kudo et al. 1993; de Boer-van Huizen and ten

Donkelaar 1999; Rose et al. 1999; Rice et al. 2010). The

lateral vestibular nucleus makes excitatory monosynaptic

connections with motor neurons of the spinal cord (Wilson

and Yoshida 1969; Grillner et al. 1970; Wilson et al. 1970;

Kasumacic et al. 2010). These vestibulospinal fibers also

have collaterals in the cervical and lower segments to

different degrees (Abzug et al. 1974; Huisman et al. 1984;

Shinoda et al. 1989; Boyle 2000). Boyle (2000) claimed

that certain vestibulospinal fibers projecting to the lumbo-

sacral cord of the squirrel monkey serve as a private

pathway to the motor circuits of the lower limb and tail

with few collaterals in the cervical cord. Other researchers

claim that descending fibers from the lateral vestibular

nucleus have ample collaterals in different segments as

demonstrated with tracer injections (Huisman et al. 1984;

Shinoda et al. 1989; Kuze et al. 1999). In the rat and cat, it

has been shown that fibers from the lateral vestibular

nucleus travel in the ventromedial funiculus (Huisman

et al. 1984; Shen et al. 1990; Rose et al. 1992, 1999;

Bacskai et al. 2002; Matesz et al. 2002) and ventrolateral

funiculus (Rose et al. 1992, 1999; Bacskai et al. 2002;

Matesz et al. 2002). These fibers mainly terminate in the

ventral horn especially in laminae 6–9 (Isu et al. 1988;

Rose et al. 1992, 1999; Bacskai et al. 2002; Matesz et al.

2002). In the mouse, spinal cord projections from the

lateral vestibular nucleus have been demonstrated with

injections of retrograde tracers into the spinal cord

(VanderHorst and Ulfhake 2006; Liang et al. 2011). How-

ever, the detailed fiber distribution of the lateral vestibular

nucleus in the mouse spinal cord is still unknown. We

examined the projections of the lateral vestibular nucleus to

the spinal cord with retrograde and anterograde tracing

techniques. We found that the lateral vestibular nucleus

projects to the gray matter at all levels in spinal cord with

fibers following both the ventral and lateral funiculi, and

terminals present in laminae 6–9. The present study

provides anatomical support for future vestibulospinal

research.

Materials and methods

Animals

Twenty-six C57/BL6 mice of 12 weeks of age, weighing

25–30 g were used. These mice were obtained from the

Animal Resource Centre in Western Australia. All exper-

imental procedures were approved by the Animal Care and

Ethics Committee of The University of New South Wales

(11/75A).

Retrograde tracing

Mice were anaesthetized with an intraperitoneal injection

of ketamine (80 mg/kg) and xylazine (5 mg/kg) and placed

in a mouse stereotaxic head holder (Kopf Instruments,

Tujunga, CA, USA). The ear bars were carefully tightened

and the mouse was stabilized in the stereotaxic holder.

After shaving the fur and sterilizing the skin, spinal cord

segments were exposed by laminectomy at C2 and T12.

The dura mater on the right side of the spinal cord was

penetrated with the tip of a 29-gauge insulin injection

needle and then the needle of a 5-ll Hamilton syringe

(Hamilton Company, Reno, NV, USA; the outer diameter

is 0.711 mm) was driven through this opening. 20–40 nl of

Brain Struct Funct

123

fluoro-gold (FG) (Fluorochrome, Denver, Co, USA; diluted

to 5 % in distilled water) solution was injected through the

needle into the right side of the spinal cord. The needle of

the Hamilton syringe was left in place for 10 min after the

injections. In the present study, ten mice were injected with

fluoro-gold into the upper cervical and upper lumbar seg-

ments (5 mice in each group). In the control group, two

mice received normal saline injections into the spinal cord

and another two mice received fluoro-gold injections into

the cisterna magna. After fluoro-gold injections, the soft

tissue and the skin were sutured and an antibiotic—tetra-

cycline (Pfizer)—was applied topically over the incision.

Buprenorphine (Temgesic, Reckitt Benckiser) solution was

injected to relieve pain.

Anterograde tracing

Mice were anaesthetized as above and positioned in the

stereotaxic instrument. After exposing the skull surface, a

hole was made dorsal to the lateral vestibular nucleus (drill

from Fine Science Tools, North Vancouver, BC, Canada).

10–20 nl of biotinylated dextran amine (BDA) solution

(10,000 MW, Invitrogen) (Bregma -5.88 to -6.24 mm,

midline ?1.25 to ?1.75 mm, surface -2.85 to -3.25 mm)

was injected into the lateral vestibular nucleus of six mice

using the Hamilton syringe described above. In the control

group, two mice received BDA injections into the cisterna

magna and another four into the adjacent vestibular nuclei

and cerebellum. The needle of the Hamilton syringe was left

in place for 10 min after BDA injections and then the skin

was sutured, buprenorphine was injected subcutaneously,

and topical tetracycline was sprayed over the incision.

Tissue preparation

Mice were anesthetized with a lethal dose of pentobarbital

solution (0.1 ml, 200 mg/ml) after 1 week (fluoro-gold

experiments) or 6 weeks (dextran experiments). They were

then perfused through the left ventricle with 60 ml of

0.9 % normal saline containing heparin (150 IU/mouse;

Sigma) using a pump. This was followed by 80 ml of 4 %

paraformaldehyde (Sigma) (in 0.1 M phosphate buffer) and

80 ml of 10 % sucrose solution (in 0.1 M phosphate buf-

fer). Mouse brain and spinal cord were removed and

postfixed in 4 % paraformaldehyde for 2 h at 4 �C, and

then transferred to 30 % sucrose (in 0.1 M PB solution).

After 48 h, brains and spinal cords were cut into 40 lm

thick sections using a Leica CM 1950 cryostat.

Immunohistochemistry

Brain and spinal cord sections from FG injected mice were

washed in 0.1 M PB and transferred to 1 % H2O2 in 50 %

ethanol at room temperature. After 30 min, the sections

were rinsed in 0.1 M PB and treated with 5 % goat serum

in 0.1 M PB to block the non-specific binding sites. The

sections were then incubated with the primary anti-FG

antibody (Chemicon, 1:5,000; raised in rabbit) overnight.

After three rinses in 0.1 M PB, the sections were incubated

with the secondary antibody (biotinylated goat anti-rabbit

IgG; Sigma, 1:200) for 2 h at room temperature. The sec-

tions were washed in 0.1 M PB and then transferred to the

extravidin peroxidase solution (Sigma, 1:1,000). After 2 h,

the sections were rinsed in 0.1 M PB and transferred to the

3,30-diaminobenzidine (DAB) reaction complex (Vector

lab, Burlingame, CA, USA) until the optimal color devel-

oped. The reaction was stopped by transferring the sections

into 0.1 M PB solution, and the sections were mounted

onto gelatinized slides and coverslipped after graded

dehydration and clearing. Sections from the dextran-

injected mouse brain and spinal cord were incubated in

1 % H2O2 in 50 % ethanol for 30 min at room temperature

and then transferred to 5 % goat serum in 0.1 M PB. These

sections were then incubated in the extravidin peroxidase

solution (Sigma, 1:1,000) for 2 h, followed by the DAB

reaction complex, before being mounted and coverslipped

as above.

Data analysis

Mouse brain sections after after visualization of fluoro-gold

injections into the spinal cord were scanned with an Aperio

scanner (ScanScope XT) under 209 magnification. Scan-

ned images were opened with Imagescope software and

images of 49 magnification were extracted and opened

with Adobe Illustrator 5. A new layer was created and then

the profile of sections was drawn. DAB-stained neurons

were mapped with purple dots. Cell numbers were calcu-

lated by counting the dots in every second section through

the entire lateral vestibular nucleus (3 series of sections

from upper cervical and upper lumbar cord injections,

respectively). For display purposes, one dot represents 3–5

labeled neurons in the diagrams. The Imagescope software

was also used to measure the nuclear diameter of labeled

neurons. The estimated cell number of lateral vestibular

neurons was calculated with the corrected Abercrombie

(1946) formula: P ¼ A MMþL. P is the estimated number of

cells, A is the actual count, M is the section thickness, and

L is the nuclear length (L was measured in every second

section). Images of 109 magnification were also extracted

from Imagescope to show the labeled neurons in the areas

of interest.

Mouse brain and spinal cord sections from dextran

injected mice were also scanned with the Aperio scanner.

Images of these sections were then extracted and overlaid

Brain Struct Funct

123

on a spinal cord diagram from the atlas of the mouse spinal

cord (Watson et al. 2009). Adjacent Nissl sections or DAB

stained sections counterstained with Nissl were referred to

in mapping. Images of 49, 59, and 109 were also

extracted to show injection sites and labeled fibers in the

brain and spinal cord.

Results

FG-labeled neurons in the lateral vestibular nucleus

After cervical cord injections of FG, labeled neurons were

first found in the vestibular complex at the level of the

facial genu. These neurons were dorsolateral to the facial

genu and ventral to the caudal part of the locus coeruleus,

occupying the ventral part of the magnocellular part of the

medial vestibular nucleus (MVe) in the mouse brain atlas

(Franklin and Paxinos 2008) (Fig. 1a, b). They were not a

homogenous group, but a mixture of large and small neu-

rons. In more caudal coronal sections, labeled neurons

were found in a wider area mediolaterally and dorsoven-

trally. They were medial to the spinal trigeminal tract and

the inferior cerebellar peduncle, leaving a narrow strip

between these two fiber bundles and the labeled neurons.

Furthermore, there was a small band devoid of labeled

neurons ventral to the superior cerebellar peduncle.

Labeled neurons formed two clusters: one large cluster in

the dorsolateral part of the vestibular complex and another

smaller cluster in the ventromedial part (Fig. 1c–e). The

majority of these neurons were large (30.3 ± 4.0 9

20.6 ± 2.2 lm) and triangular or fusiform. Medial to the

ventromedial cluster, there were some labeled neurons

scattered in the medial vestibular nucleus (MVe). They

were also large neurons. In the caudal pole of the lateral

vestibular nucleus, labeled neurons seemed to form only

one cluster and other labeled neurons were sparsely dis-

tributed in the ventral part of the lateral vestibular nucleus,

Fig. 1 Retrogradely labeled neurons after FG injections into the

cervical cord. a–f Diagrams showing FG-labeled neurons in the

vestibular complex. a0–f0 Photographs showing FG-labeled neurons in

the vestibular complex. From the rostral pole of the lateral vestibular

nucleus to the caudal pole, the number of labeled neurons increases

(a–d, a0–d0) and then wanes (e–f, e0–f0), with a dense cluster of

neurons in the dorsal part of the nucleus and a less dense cluster in the

ventral part. Some labeled neurons are diffusely distributed in the

caudoventral part of the vestibular complex, which might correspond

to the ventral part of the lateral vestibular nucleus. Note that a small

number of labeled neurons are present in the medial part of the

vestibular complex, which corresponds to the medial vestibular

nucleus. The green dashed lines indicate the SuVe boundary, the lightblue dashed lines indicate the MVe boundary and the red dashed linesindicate the LVe boundary. The photomicrograph in a shows the

injection site in the mouse cervical cord. The scale bar for all

photographs is 200 lm

Brain Struct Funct

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which may correspond to the ventral part of the lateral

vestibular nucleus (Fig. 1f). In total, there were 660 ± 32

FG-labeled neurons.

After FG injections at lumbar cord levels, labeled neu-

rons were also found in the rostral pole of the vestibular

nucleus and they were also dorsolateral to the facial genu

and ventral to the caudal part of the locus coeruleus

(Fig. 2a, b). In more caudal sections, labeled neurons were

found in the more lateral and dorsal parts of the lateral

vestibular nucleus (Fig. 2c–f). These neurons shared similar

features to those found after cervical cord injections of FG

except that there were fewer labeled neurons after lumbar

cord injections (425 ± 21). In the caudal pole of the lateral

vestibular nucleus, labeled neurons were also packed into a

cluster with a few neurons scattered in the ventral part of the

vestibular complex, which corresponds with the ventral part

of the lateral vestibular nucleus (Fig. 2f).

BDA-labeled fibers and boutons in the spinal cord

After BDA injections to the lateral vestibular nucleus

(Fig. 3a), anterogradely labeled fibers were seen to travel

ventromedially toward the midline where they joined the

medial longitudinal fasciculus (mlf) on the ipsilateral side.

Some fibers crossed the midline and travelled in the con-

tralateral mlf (Fig. 3b). These contralateral fibers joined the

mlf at a more rostral level than their ipsilateral counterpart

(Fig. 3c). These fibers remained in the mlf until the caudal

pole of the hindbrain. It should be noted at this stage that

while our target was the lateral vestibular nucleus,

involvement of the lateral part of the medial vestibular

nucleus cannot be excluded.

In the white matter of the spinal cord, labeled fiber tracts

were seen on both sides. Ipsilateral fiber tracts were located

in the ventral funiculus and ventrolateral part of the lateral

funiculus at all spinal cord levels. Within the ventral

funiculus, the greatest labeling was in its medial part where

fibers were packed and close to the ventral surface of the

spinal cord. In the rest of the ventral funiculus at cervical

and thoracic segments, fibers were distributed more dif-

fusely (Fig. 3d). In some segments, fibers were also seen in

the dorsolateral part of the lateral funiculus. Contralateral

fiber bundles were fewer in number and they were located

in the ventral funiculus (Fig. 3d). However, these contra-

lateral fiber bundles were not densely packed as their

ipsilateral counterparts and they were only found in the

Fig. 2 Retrogradely labeled neurons after FG injections into the

lumbar cord. a–f Diagrams showing FG-labeled neurons in the

vestibular complex. a0–f0 Photographs showing FG-labeled neurons in

the vestibular complex. Similar to the pattern seen after cervical cord

injections, the number of labeled neurons increases (a–e, a0–e0) and

wanes from rostral to caudal (f, f0), with a dense cluster of neurons in

the dorsal part of the LVe. Some labeled neurons are diffusely

distributed in the caudoventral part of the vestibular complex, which

we believe to be the ventral part of the lateral vestibular nucleus. No

labeled neurons are present in the medial part of the vestibular

complex. The photomicrograph in a shows the injection site in the

mouse lumbar cord. The scale bar for all photographs is 200 lm

Brain Struct Funct

123

cervical and upper two-thirds of the thoracic cord. We

believe that these contralateral fibers might arise from

the medial vestibular nucleus. In the present retrograde

labeling study, there were no labeled neurons in the

medial vestibular nucleus after lumbar cord injections of

FG and this may explain the termination of fibers from

the medial vestibular nucleus only in cervical and thoracic

segments. The other possibility is that the lateral vestib-

ular nucleus innervates both sides of the cervical and

thoracic cord.

After injections of BDA into the lateral vestibular

nucleus, labeled fibers and boutons were widely distributed

in the ipsilateral laminae 6–10 in the gray matter of the

spinal cord, especially in the medial two-thirds of the gray

matter in the entire spinal cord (Fig. 3d, e, g–i). However,

the density of labeled fibers and boutons was highest in

lamina 8. In lamina 9, many fibers were seen to terminate

on the medial group of motor neurons in the ventral horn.

In the lateral part of lamina 9, labeled fibers were mainly

found in the medial part of the motor neuron groups that

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innervate the distal limb muscles (Fig. 3e, g, i). In both the

medial and lateral motor neuron groups, many swellings

presumed to be boutons were seen adjacent to motor neu-

rons (Fig. 3e). In lamina 10, fibers and boutons were pre-

dominantly seen in the area ventral and lateral to the

central canal (CC). Labeled fibers in the dorsal horn were

occasionally seen. In the contralateral gray matter of the

spinal cord, there were fewer labeled fibers and they were

distributed in the gray matter in a pattern similar to that of

ipsilateral fibers, but with a lower density (Fig. 3d, f, g).

These contralateral fiber tracts were only seen in cervical

and upper two-thirds of the thoracic spinal cord. Some

contralateral fibers were also seen in lower lumbar seg-

ments (Fig. 3h). These fibers were sparsely distributed in

laminae 6–8. We believe that they must be the collaterals

of the ipsilateral vestibulospinal fiber tract that crossed the

midline, because there were no labeled fiber bundles on the

contralateral side at these levels.

When BDA was injected into the cisterna magna or

cerebellar cortex, no labeled fibers were found in the spinal

cord. Small injections to the dorsal part of the lateral

vestibular nucleus mainly labeled fibers in the dorsolateral

funiculus (Fig. 4a–c). Large injections (Fig. 4d), which

involved the lateral part of the medial vestibular nucleus

and the superior vestibular nucleus as well as the lateral

nucleus, revealed a pattern of fiber distribution similar to

Fig. 3 continued

Fig. 3 Anterogradely labeled fibers after BDA injections into the

vestibular complex. a The injection site in the vestibular complex,

predominantly in the lateral vestibular nucleus. The dashed-linecircled area is the injection site. b BDA-labeled fiber tracts in the

contralateral medial longitudinal fasciculus at the level of the caudal

pole of the facial nucleus (arrows). c BDA-labeled fibers in bilateral

medial longitudinal fasciculus at the level of the rostral pole of the

hypoglossal nucleus (arrows). d A diagram of BDA-labeled fibers in

the sixth cervical segment. On the ipsilateral side, labeled fiber tracts

are present in the ventral and ventrolateral funiculi (red circled area).

Fibers and boutons are found in laminae 6–10 with predominance in

lamina 8. On the contralateral side, labeled fiber tracts are present in

the ventral funiculus only (black circled area) and they are diffusely

distributed. Labeled fibers and boutons are mainly present in laminae

7 and 8, but the density of these labeled fibers is lower than that of the

ipsilateral fibers. The majority of the fibers are in laminae 7 and 8. e A

higher magnification image of the right rectangular area in

d. Labeled fibers and boutons are present in laminae 8 and 9

(arrows). Some boutons are adjacent to motor neurons (dashed linecircled neurons) in the medial motor neuron group. Note the larger

magnification of the small rectangular area, which shows the boutons

terminating on the cellular area of a motor neuron at the upper rightcorner (arrow). f A higher magnification image of the left rectangular

area in d. Labeled fibers and boutons are present in laminae 7–9

(arrows). g A photograph showing BDA-labeled fiber tracts and

terminals in the seventh thoracic cord segment. Ipsilaterally labeled

fibers are present in both the ventral and ventrolateral funiculi (the redcircled area). The contralaterally labeled fibers are present in the

ventral funiculus only, with an even lower density than their

counterparts in the cervical cord. In the gray matter, fibers and

boutons are distributed similarly as those in the cervical segments

(arrows). h A photograph showing BDA-labeled fiber tracts and

terminals in the sixth lumbar segment. Fiber tracts are only present in

the ipsilateral ventral and ventrolateral funiculi (red circled area).

Fibers and boutons are present in laminae 6–9 with predominance in

lamina 8. A higher magnification image of the rectangular area is

shown in i (arrows). Note that a few fibers are present in the

contralateral gray matter. We believe that they arise from the

ipsilateral tracts. The scale bar for a, g, and h is 200 lm; the scalebar for b, c, and i is 200 lm; the scale bar for d is 500 lm; the scalebar for e and f is 50 lm. The scale bar for the photomicrograph in

d is 25 lm

b

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that described above. However, the contralateral fiber tracts

were more widely spread in the ventral funiculus and their

fibers mainly terminated in the medial third of the gray

matter in laminae 7–9 (Fig. 4e). On the ipsilateral side,

densely packed vestibulospinal fibers (more ventrally

located) were mixed with the diffusely distributed fibers in

the ventral funiculus (Fig. 4e). In the lumbar and sacral

segments, only ipsilateral vestibulospinal fibers were seen.

These fibers were located in the ventral and lateral funiculi

(Fig. 4f).

Discussion

Boundaries of the lateral vestibular nucleus defined

by retrograde tracing

The lateral vestibular nucleus has historically been defined

by the presence of very large neurons. This nucleus extends

from the level of the rostral pole of the dorsal cochlear

nucleus to the level of the caudal nucleus Y (Watson 2012).

In the present retrograde labeling study, FG-labeled

neurons in the lateral vestibular nucleus were found ros-

trally in the ventral part of the vestibular complex at the

level of the facial genu after cervical and lumbar cord

injections of FG. In the caudal part of the nucleus, the

number of labeled neurons was greater, and there were

labeled neurons in the dorsal part of the nucleus as well. At

the level of the caudal pole of nucleus Y, labeled neurons

were only found in the area medial to nucleus Y and there

was a higher density of labeled neurons (Figs. 1d0, e0, 2d0,e0). Labeled neurons in the ventral part of the lateral ves-

tibular nucleus were intermingled with large fiber bundles.

Some labeled neurons were found in the medial vestibular

nucleus that is medial to the lateral vestibular nucleus,

forming a wedge shape with the tip toward the 4th ven-

tricle. The present study was consistent with findings of

previous studies (in mice: VanderHorst and Ulfhake 2006;

in rats: Zemlan et al. 1979; Leong et al. 1984; in cats:

Hayes and Rustioni 1981). However, the topography of the

vestibulospinal neurons was not consistent with that

observed as in the cat (Hayes and Rustioni 1981).

This study has raised doubts about the exact border

between the medial and the lateral vestibular nuclei.

Fig. 4 a An injection site in the dorsal part of the lateral vestibular

nucleus (blue circled area). b BDA-labeled fibers in the fourth

cervical segment. Note that more fiber tracts are present in the

ipsilateral dorsolateral funiculus (blue circled area) than in the ventral

funiculus. Fiber terminals are mainly present in laminae 7 and 8. No

fibers are found in the contralateral cord. c BDA-labeled fibers in the

third lumbar segment. Note that the termination pattern of labeled

fibers and boutons in the lumbar cord is similar to that in the cervical

cord. d An injection site in the vestibular complex which involves the

medial vestibular nucleus. e BDA-labeled fibers and boutons in the

fifth cervical segment. Note the large number of labeled fiber tracts

(black circled area) in the contralateral ventral funiculus. The

contralateral fibers terminate mainly in the medial third of the gray

matter (arrows on the left). The distribution of the ipsilateral fibers

and boutons is similar to that seen in Fig. 3d–f. f BDA-labeled fibers

and boutons in the fourth lumbar segment. Fiber tracts are only

present in the ipsilateral ventral and ventrolateral funiculi (red circledarea), but fiber terminals are present bilaterally in the gray matter

with a small number of fibers in the contralateral cord. In the

ipsilateral gray matter, a small number of fibers are also seen in

lamina 5 (arrows). The scale bar for a–f is 200 lm

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Paxinos and Watson (1982) in their rat brain atlas defined a

larger lateral vestibular nucleus at the expense of the

medial vestibular nucleus. In the 2008 edition of the mouse

brain atlas (Franklin and Paxinos 2008), as well as in the

more recent edition of the mouse brain atlas (Paxinos and

Franklin 2013), the medial vestibular nucleus was made

larger at the expense of the lateral vestibular nucleus.

Because immunohistochemical stains (Watson and Paxinos

2010) and in situ data (i.e., Allen Brain Atlas,

http://mouse.brain-map.org) do not reveal a distinct

boundary between the two nuclei, it is hard to know where

the true borders might lie. Some have argued that the medial

vestibular nucleus does not project below the thoracic cord

(Wilson et al. 1967). By that criterion, all that is currently

delineated in the atlas of the medial vestibular nucleus

should be transferred to the lateral vestibular nucleus (col-

ored dashed lines in Fig. 1) since these neurons project to

the lumbar spinal cord. Certainly the magnocellular part of

the medial vestibular nucleus has larger neurons than the

parvicellular part of the medial vestibular nucleus, but the

neurons of the magnocellular part of the medial vestibular

nucleus are not as large as those in the adjacent area which

the atlases label the lateral vestibular nucleus.

As noted above, the currently available data from

immunohistochemistry, in situ hybridization, and Nissl

staining do not adequately define the precise boundary

between the lateral vestibular nucleus and the medial

vestibular nucleus. We believe that the present tracing

study might be used to define a new boundary between

these nuclei, and the validity of this boundary could be

tested in future electrophysiological studies (Fig. 5).

It has been reported that some vestibulospinal neurons in

the lateral vestibular nucleus are glutamic acid decarbox-

ylase (GAD) positive (Valla et al. 2003). Whereas in

another study, 97 % of the vestibulospinal fibers inner-

vating the lumbar cord have been found to be vesicular

glutamate transporter 2 (vGluT2) positive (Du Beau et al.

2012), indicating that these vestibulospinal fibers are

excitatory. In the mouse, neurotransmitters used by the

vestibulospinal neurons have not been investigated.

Fig. 5 A summary diagram showing the origin, course and termina-

tion of the descending fibers from the lateral vestibular nucleus to the

spinal cord. Vestibulospinal fibers originate in the lateral vestibular

nucleus and descend in the hindbrain either within the ipsilateral

medial longitudinal fasciculus or lateral to it. These descending fibers

travel in the ipsilateral ventral and lateral funiculi in the white matter

of the spinal cord and terminate in laminae 6–10, with predominance

in laminae 8 of the ipsilateral side. A small number of fibers were

occasionally found in ipsilateral lamina 5. Contralateral collaterals

were present in the lumbosacral cord. It was uncertain whether

collaterals were also present in the cervical and thoracic segments.

Therefore, only collaterals in the lumbar cord were represented in this

diagram

b

Brain Struct Funct

123

Fiber termination of the lateral vestibular nucleus

in the spinal cord

We believe that the present study is the first to describe

the fiber termination of the lateral vestibular nucleus in the

spinal cord of the mouse. The fibers projecting from the

lateral vestibular nucleus travelled in the ipsilateral ventral

and lateral funiculi and terminated in the ventromedial gray

matter of the ipsilateral spinal cord. In some lumbar and

sacral cord sections, collaterals of vestibulospinal fibers

were found in the contralateral cord. In the cervical and

upper thoracic cord, some fiber tracts and terminals were

also found in the contralateral cord. We think it is likely that

these fibers have arisen from the medial vestibular nucleus.

In the rat, it has been reported that vestibulospinal tracts

travel in the ipsilateral ventral funiculus, and to a lesser

extent in the ipsilateral lateral funiculus. Their fibers

mainly terminate in the ventral horn, especially in laminae

7 and 8, and to a lesser extent in the dorsal horn. Fur-

thermore, a considerable number of collaterals are also

present in the cervical and lumbosacral segments (Bacskai

et al. 2002; Matesz et al. 2002). Our results are consistent

with the findings in the rat.

In the cat (Rose et al. 1999), fibers from the lateral

vestibular nucleus have been traced to the upper cervical

cord where they are bilaterally distributed in the ventro-

medial funiculus, but only ipsilaterally in the ventrolateral

and lateral funiculi. This finding is similar to ours in

relation to the distribution of vestibulospinal fibers in the

cervical cord. However, these authors did not investigate

the fiber termination in lower spinal segments. Another

study in the cat (Kuze et al. 1999) revealed the lateral

vestibular nucleus fiber distribution in the entire spinal cord

by injecting PHA-L into the lateral vestibular nucleus.

Vestibulospinal fibers were seen to travel in the ventral

funiculus bilaterally in the cervical cord, but only in the

ipsilateral ventral funiculus in lower segments. This result

is consistent with our findings.

In the squirrel monkey (Boyle 2000), vestibulospinal

fibers from the lateral vestibular nucleus were seen to reach

the lumbar cord with few collaterals in the cervical cord.

Boyle concluded that these lumbar projecting fibers may

serve as a ‘private pathway’ connecting the lateral vestib-

ular nucleus neurons and the lumbar cord.

In the present study, some fibers were also found in the

contralateral lumbar and sacral cord, but no contralateral

tract was observed. These contralateral fibers must there-

fore originate from the ipsilateral vestibulospinal tract.

This phenomenon was also observed in an electrophysio-

logical study, which showed that the lumbar cord was

innervated by the lateral vestibulospinal tract only, and that

there are bifurcating fibers innervating the contralateral

spinal cord (Kasumacic et al. 2010).

In this study, a few fibers were found in the dorsolateral

funiculus after injecting BDA to the dorsal part of the

lateral vestibular nucleus. These fibers are apparently dif-

ferent from those in the ventral funiculus. Based on their

location in the vestibular complex and their discrete char-

acter, we propose that they might arise from the superior

vestibular nucleus.

Functional significance of spinal projections

from the lateral vestibular nucleus

The present study has clearly shown that fibers from the

lateral vestibular nucleus innervate not only the axial motor

neurons, but also the motor neurons in the lateral motor

neuron group and interneurons in laminae 7 and 8. This is

consistent with previous findings that the lateral vestibular

nucleus is involved in both posture and balance control. The

contralateral extensor motor neurons and bilateral flexor

motor neurons might be activated and inhibited, respec-

tively, through commissural interneurons as found in other

mammals (Wilson and Yoshida 1969; Pompeiano 1972).

It has been shown that somatosensory and vestibular

inputs converge on interneurons in the spinal cord (Brink

et al. 1980). These interneurons might mediate various

responses to external stimuli through their connections

with other neurons in the spinal cord. For example, inter-

neurons in laminae 7 and 8 of the spinal cord may partic-

ipate in locomotion through connections with the central

pattern generator (Kjaerulff et al. 1994; Bracci et al. 1996;

Tresch and Kiehn 1999; Cina and Hochman 2000; Dai

et al. 2005). It is highly possible that vestibulospinal fiber

terminals on the interneurons in laminae 7 and 8 are indi-

rectly engaged in locomotion.

Conclusion

The present study revealed in the spinal cord the distribu-

tion pattern of fibers from the lateral vestibular nucleus and

proposed the boundary between the lateral and the medial

vestibular nuclei based on retrograde tracing results. This

can provide an anatomical foundation for further vestibular

research on the mouse.

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