Expression of Calcitonin Gene-Related Peptide in
Efferent Vestibular System and Vestibular Nucleus in
Rats with Motion Sickness
Wang Xiaocheng1, Shi Zhaohui2, Xue Junhui1, Zhang Lei1, Feng Lining1*, Zhang Zuoming1*
1Department of Clinical Aerospace Medicine, Key Laboratory of Aerospace Medicine of Ministry of Education, The Fourth Military Medical University, Xi’an, China,
2Department of Otolaryngology Head and Neck Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an, China
Motion sickness presents a challenge due to its high incidence and unknown pathogenesis although it is a known fact that a
functioning vestibular system is essential for the perception of motion sickness. Recent studies show that the efferent
vestibular neurons contain calcitonin gene-related peptide (CGRP). It is a possibility that the CGRP immunoreactivity (CGRPi)
fibers of the efferent vestibular system modulate primary afferent input into the central nervous system; thus, making it
likely that CGRP plays a key role in motion sickness. To elucidate the relationship between motion sickness and CGRP, the
effects of CGRP on the vestibular efferent nucleus and the vestibular nucleus were investigated in rats with motion sickness.
Methods: An animal model of motion sickness was created by subjecting rats to rotary stimulation for 30 minutes via a
trapezoidal stimulation pattern. The number of CGRPi neurons in the vestibular efferent nucleus at the level of the facial
nerve genu and the expression level of CGRPi in the vestibular nucleus of rats were measured. Using the ABC method of
immunohistochemistry technique, measurements were taken before and after rotary stimulation. The effects of
anisodamine on the expression of CGRP in the vestibular efferent nucleus and the vestibular nucleus of rats with motion
sickness were also investigated.
Results and Discussion: Both the number of CGRPi neurons in the vestibular efferent nucleus and expression level in the
vestibular nucleus increased significantly in rats with motion sickness compared to that of controls. The increase of CGRP
expression in rats subjected to rotary stimulation 3 times was greater than those having only one-time stimulation.
Administration of anisodamine decreased the expression of CGRP within the vestibular efferent nucleus and the vestibular
nucleus in rats subjected to rotary stimulation. In conclusion, CGRP possibly plays a role in motion sickness and its
mechanism merits further investigation.
Citation: Xiaocheng W, Zhaohui S, Junhui X, Lei Z, Lining F, et al. (2012) Expression of Calcitonin Gene-Related Peptide in Efferent Vestibular System and
Vestibular Nucleus in Rats with Motion Sickness. PLoS ONE 7(10): e47308. doi:10.1371/journal.pone.0047308
Editor: Bruce Riley, Texas A&M University, United States of America
Received May 1, 2012; Accepted September 11, 2012; Published October 9, 2012
Copyright: ? 2012 Xiaocheng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Shaanxi Natural Science Fund 2009K17-02(46), China. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (FL); firstname.lastname@example.org (ZZ)
Motion sickness is a common and challenging problem. Various
physiological measurements for this problem have been tested.
However, no single parameter has yet been found to have a high
enough sensitivity and specificity for the diagnosis or prediction of
individual susceptibility to motion sickness. [1–3] Motion sickness
may be precipitated by conflicting sensory input – visual and
vestibular signals that do not match an internal model of expected
environmental stimuli. [1–4] It is well known that a functioning
vestibular system is essential for the perception of motion sickness.
 Theoretically, if sufficiently provocative motion stimulus is
introduced, anyone with a functioning vestibular system could be
susceptible.  However, thus far the underlying mechanism is
unclear. The innervations of the vestibular system include both the
afferent and efferent vestibular system (EVS). Vestibular sensory
organs in the inner ear are innervated by true efferent fibers
originating from brainstem neurons. Studies show that electrical
stimulation of EVS fibers can result in both facilitatory and
inhibitory modulation of the sensory activity in the afferent
vestibular system.  Therefore, EVS is considered to play a role
in the modulation of the afferent input from the peripheral
vestibular receptors to the central nervous system. 
Originally, efferent vestibular neurons (EVN) were assumed to
be cholinergic, but currently, more evidence demonstrates that the
efferent vestibular neurons contain both calcitonin gene-related
peptide (CGRP) and choline acetyltransferase (CHAT). CGRP is a
peptide with 37 amino acid residues translated from alternative
processing of mRNA transcribed from the calcitonin gene. [8–10]
CGRP is widely distributed in the central nervous system
including the vestibular pathways. CGRP can be detected in the
efferent pathways of the vestibular end-organs and the central
vestibular system. [7,11–13] Therefore, the role of CGRP in
modulating this afferent input into the central nervous system is of
fundamental importance in understanding neural processing in
general and in the etiology of motion sickness. However, at
PLOS ONE | www.plosone.org1October 2012 | Volume 7 | Issue 10 | e47308
present, much effort has been directed toward the understanding
of the mechanism of CHAT in motion sickness, and anticholin-
ergics are the most commonly used pharmacological agents today
for prevention and treatment of this problem.  It is, therefore,
necessary to explore the relationship between CGRP and the
vestibular system and the role of CGRP in motion sickness.
We hypothesize that the EVS plays a role in the process of
motion sickness via CGRP. To the best of our knowledge, there is
little information about this. In this study, we establish an animal
model of motion sickness in rats by rotary stimulation for 30
minutes in a trapezoidal stimulation pattern. We then measure
and compare the number of CGRPi neurons in the vestibular
efferent nucleus (VEN) of the brainstem at the level of the genu of
the facial nerve and the level of expression of CGRP immuno-
reactivity in the vestibular nuclei of the brainstem. The
measurements were taken before and after rotary stimulation by
utilizing the immunohistochemistry technique. The effect of
anisodamine (an anticholinergic) on the expression of CGRP
within the vestibular efferent nucleus and the vestibular nucleus
were also investigated. This study attempts to present a promising
new direction in exploring the mechanism, prevention, and
treatment of motion sickness.
Materials and Methods
All experiments used white, male Sprague-Dawley (SD) rats
weighing about 220 g each. All animal procedures described in
this study were performed in adherence with the Guide for the
Care and Use of Laboratory Animals published by the US
National Institutes of Health (NIH Publication No. 85–23, revised
1996) with approval from the Committee on the Ethics of Animal
Experiments of the Fourth Military Medical University. All
surgery was performed under diethyl ether anesthesia, and all
efforts were made to minimize suffering.
Establishment of an animal model of motion sickness
Rotary stimulation equipment and method.
animal centrifuge unit (Yongdao Medicine Instrument Company,
Japan) was used to create an animal model of motion sickness. The
unit is composed of a generator and an arm with two suspended
plexiglass cages. The radius from the center of rotation to the point
of suspension of the cages is 0.6 m. The angular acceleration,
angular velocity, and run-time are controlled by computer. The
cages not only revolve around a vertical axis but also can move
along the direction of the arm during rotation. The rats can move
around freely in the cages.
The cages were accelerated at 100/s2to a peak speed of 240u/s
and rotated at peak speed for 5 minutes. Then the cages were
decelerated at 10u/s2to 0u/s. After that, the clockwise rotation
alternated with the counterclockwise rotation. This stimulation
lasted 30 minutes. Thus, the rats were treated with 1.46 G gravity
force for 5 minutes – +10u/s2angular acceleration, 10 s and
210u/s2angular acceleration, 10 s tautologically, and 0.41/s2
Coriolis cumulative acceleration in each stimulation. 
Verification of an animal model of motion sickness by
conditioned taste aversion.
Conditioned taste aversion (CTA)
tests were performed to verify that the animal model of motion
sickness induced by the rotary stimulus was valid. Forty (40) rats
were divided into 4 groups (10 per group) using a random digit
table (Table 1). The 4 groups were: A) rotary stimulation undosed;
B) rotary stimulation saline dosed; C) rotary stimulation anisoda-
mine dosed; D) control group. Rats in groups A, B, and C were all
subjected to rotary stimulation for 30 minutes. Thirty (30) minutes
before rotary stimulation, group C was administered anisodamine
orally (0.1 mg/100 g body weight) and group B was administered
saline orally (0.1 mg/100 g body weight).  The control group
had no rotary stimulation.
After rotary stimulation, all 4 groups were given water with
0.15% saccharin. The intake volume of saccharin solution was
measured every 24 h. The volumes consumed in the first 24 h,
second 24 h, and third 24 h after stimulation were compared with
the volume in the 24 h prior to the stimulation. 
Fifty (50) rats were divided into 5 groups (10 per group) using a
random digit table (Table 2). The 5 groups were: I) triple rotary
stimulation undosed; II) single rotary stimulation undosed; III)
single rotary stimulation saline dosed; IV) single rotary stimulation
anisodamine dosed; V) control group. Rats in group I were
subjected to rotary stimulation 3 times at 24 h intervals, and
groups II, III, and IV were subjected to rotary stimulation once.
The control group was not subjected to rotary stimulation. Thirty
(30) minutes before rotary stimulation, rats in group IV were
administered anisodamine (0.1 mg/100 g body weight) orally and
rats in group III were administrated saline (0.1 mg/100 g body
Rats were anesthetized with diethyl ether after undergoing
single or triple rotary stimulation. Cardiac perfusions were
performed with phosphate buffered saline (200 ml, pH 7.2,
5 min) followed by 4% paraformaldehyde in 0.1 M phosphate
buffer (500 ml, pH 7.4, 20 min). The brain was then removed
and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer
(pH 7.4) for 4 h before being stored overnight (4uC) in 0.1 M
phosphate buffer (pH 7.4) containing 30% sucrose. The next
day, transverse serial sections (30 mm) were sliced through the
brainstem from the hypoglossal nucleus to the anterior aspect of
the parabrachial nucleus on a sliding microtome (CM1900
cryostat manufactured by Leica, Germany) and collected in
0.1 M phosphate buffer (pH 7.4, 4uC). Alternate sections were
Table 1. Experimental groups for verifying the animal model
of motion sickness.
grouprotary stimulationsaline dosedanisodamine dosed
Table 2. The experimental groups for
grouprotary stimulationsaline dosed
CGRP in Vestibular System of Rats with MS
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collected and divided into 2 groups. The tissue sections from the
first group were immunohistochemically labeled for CGRP, and
10–20 sections were incubated in plastic boats with rabbit anti-
CGRP (diluted 1:1,000, Chemico Company, Ltd, Malaysia)
overnight at 4uC. Antibody-antigen binding was visualized using
an avidin-biotin-peroxidase complex (ABC Kit, Vector Labo-
ratories, America) with 3, 30-diaminobenzidine tetrahydrochlor-
ide (Sigma-Aldrich Chemicals, America) as the chromogen.
After the reaction, brainstem sections were mounted on chrome
alum-gelatin-coated microscope slides, air-dried, dehydrated in
ethanol, cleared in xylene, and cover-slipped with Permount.
To ensure specificity of the CGRP staining procedure, the tissue
sections from the second group were incubated in goat blood
serum instead of rabbit anti-CGRP. The visualization steps were
the same as for the first group. No immunoreactive signal was
observed in these sections, indicating that any observed immuno-
reactivity was due to CGRP and not non-specific binding.
All tissue sections were examined with an Olympus Vanox-T
microscope using bright field illumination. The vestibular efferent
nucleus, as described by Tanaka et al. and Wackym et al., and the
vestibular nucleus were studied. [18,19] The cell bodies of CGRP
positive and CGRPi fibers were identified and photographed.
The dorsolateral to the genu of the facial nerve (DL), the medial
to the genu of facial nerve (M), and the caudal pontine reticular
nucleus (CPR) regions were examined for CGRP labeled cells.
After initial evaluation, labeled cells in DL, M, and CPR regions
were counted using a Leica Q-500 microscope fitted with image
analysis hardware and software for densitometry and particle
analysis (Microcomp Image Analysis System, Southern Micro
Instruments, Inc., Atlanta GA). Then, the average background
gray levels were determined for each stained batch of sections.
Pixels with gray levels at least 10% darker than the average
background levels were defined as positive for CGRP labeling.
This resulted in a two-level image (black/white) that was further
processed to identify contiguous pixels so that neuronal cell bodies
could be automatically recognized and counted.  Gray scale of
CGRP fibers in the vestibular nucleus was also quantitatively
analyzed. Five (5) HP visual fields in the vestibular nucleus were
selected to evaluate the optical density of CGRP fibers in each
section. The optical density of CGRP immunohistochemistry in
the vestibular nucleus was calculated as the optical density
observed minus the background density.
Image statistical analysis
Data is expressed as a mean6S.D. All statistical analyses were
done using the SPSS 8.0 statistical software package. The intake
volumes of 0.15% saccharin solution before and after stimulation
within each group were statistically analyzed with univariate
ANOVA. The reductions in intake volumes after stimulation were
calculated as the volume after stimulation minus the volume before
stimulation in the 4 groups and the differences between the groups
were also compared with univariate ANOVA. P values,0.05 were
Five (5) tissue sections were selected from each rat after
immunohistochemistry. The number of CGRP positive cells and
optical density were statistically analyzed with univariate ANOVA.
Statistical significance of the differences between groups was
evaluated using the Student-Newman-Keuls test.  Significance
was assigned at the P,0.05 level.
Rotary stimulation and conditioned taste aversion
The average intake volume of saccharin solution was 64.0 ml
before rotary stimulation in group A, while the average intake
volumes were 38.4 ml, 41.6 ml, and 43.3 ml in the first 24 h,
second 24 h, and third 24 h after stimulation, respectively
(Table 3). In group A, the intake volumes in the first 24 h, second
24 h, and third 24 h after stimulation were 60.0%, 64.9%, and
67.8%, compared with that before stimulation. Results of such
comparisons were 62.1%, 65.8%, and 68.4% in group B; and
81.1%, 84.6%, and 84.8% in group C, respectively. Saccharin
solution intake after the rotary stimulation was reduced in groups
A, B and C compared to that prior to the stimulation (Figure 1).
The differences were significant in the 3 groups (P,0.05).
Although the intake volumes were reduced in the 3 groups after
stimulation, the reduction in group C was significantly less than
those of group A and group B in the three 24 h intervals. The
reductions show no significant difference between group A and
group B in the 3 24 h intervals. Animals from group D showed no
significant intake volume change during the experimental period.
Expression of CGRP in the vestibular efferent nucleus and
the vestibular nucleus
The CGRP-positive EVN were located in the brainstem and
were small fusiform-shaped neurons mainly composed of 3 groups
of neurons: neurons dorsolateral to the genu of the facial nerve
(DL); neurons dorsomedial to the genu of facial nerve (M); and
scattered cells throughout the caudal pontine reticular nucleus
(CPR). The average number of CGRPi neurons in the vestibular
efferent nucleus of the brainstem were 16.86, 10.13, 10.28, 7.25,
and 6.00 in groups I, II, III, IV, and V, respectively. The number
of CGRPi neurons in the vestibular efferent nucleus increased
significantly in groups I and II compared with group V (P,0.05).
The increase of CGRPi neurons in group I was significantly
greater than that in group II. The number of CGRPi neurons in
the vestibular efferent nucleus was significantly decreased in
group IV compared with that in groups I, II, and, III (P,0.05).
There was no significant difference between groups II and III
(Figures 2, 3).
CGRPi fibers were observed in the vestibular nucleus (Figures 4,
5). The average optical density of CGRP immunohistochemistry
in the vestibular nucleus were 81.1, 52.9, 54.2, 41.8, and 36.7 in
groups I, II, III, IV, and V, respectively. The level of
immunoreactivity in the vestibular nucleus increased significantly
in groups I and II compared to group V (P,0.05). The increase in
group I was greater than that in group II. In group IV, the level of
such immunoreactivity in the vestibular nucleus was significantly
decreased compared to groups I, II, and III. There was no
significant difference between groups II and III.
Discussion and Conclusion
Prevention and treatment of motion sickness is a challenge,
particularly in aerospace medicine, due to its high incidence and
unclear pathogenesis. At present, animal models of motion
sickness have been developed in cats, dogs, rats, and squirrel
monkeys. CTA and pica are classic indices to evaluate the animal
model of motion sickness. CTA, a significant decrease in the
animals’ consumption of some substance with a certain taste (e.g.,
saccharin solution), can be induced by various stimuli. Pica, an
increase in the animals’ consumption of kaolin or other substances
of no nutritional value, can also be induced. Today, CTA is
extensively used to evaluate the animal model of motion sickness
CGRP in Vestibular System of Rats with MS
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by observing the decrease in consumption of saccharin solution
after stimulation, while pica is used by observing the increase in
consumption of kaolin after stimulation. Recent studies suggest
other new indices for the study of motion sickness by observing
symptoms after rotation, such as piloerection, tremble, urinal and
fecal incontinence. However, the utility and broad acceptance of
these newer indices may require further investigation. 
Compared to observing symptoms and Pica, CTA is sensitive,
simple, stable, easy to perform, and applicable to a variety of
animals. Furthermore, CTA is readily quantifiable and, conse-
quently, more commonly used. Moreover, CTA is a behavioral
index that, through the degree of antidipsia, reflects the severity of
motion sickness. [14,21] Therefore, in our experiments, we
observe the changes in the intake volumes of saccharin solution
before and after rotation to evaluate the animal model of motion
sickness in rats. The intake volume of 0.15% saccharin solution
was significantly reduced after motion stimulation. Antidipsia can
be decreased by administration of anisodamine – an anticholin-
ergic drug currently used to prevent and treat motion sickness.
Observation of this effect further indicates the validity of our
animal model using rotary stimulation for 30 minutes in a
The efferent vestibular neurons (EVN) are located in the
vestibular efferent nucleus (VEN) and are mainly composed of 3
types of neurons – DL, M, and CPR. Many of the EVN are
CGRPi and send efferent fibers to the vestibular end-organs.
[13,22,23] In our experiments, the number of CGRPi neurons in
VEN increased significantly in rats after rotary stimulation.
Moreover, the increase of CGRPi neurons in rats after rotary
stimulus was 3 times greater than that of the rats that underwent
rotary stimulus only once.
The exact role of CGRP in EVS still is unclear today, but it has
been found that CGRP increases the discharge firing rate of
afferent fibers innervating the hair cells in the lateral line organ of
Xenopus laevis.  In end-organs of the human vestibule,
CGRPi is located in vesiculated nerve fibers and bouton-type
nerve terminals that directly contact afferent nerve chalices
surrounding type I sensory cells and afferent nerve fibers to form
an en passant contact with afferent dendrites. [16,22] It follows that
the release of CGRP is able to directly alter primary afferent
inputs via type I hair cells of the central vestibular nervous system.
On the other hand, EVN contain both CGRP and choline
acetyltransferase; and studies have found an interaction between
CGRP and acetylcholine. [16,22,23] The acetylcholine-mediated
efferent system is thought to provide a tonic inhibitory influence
Figure 1. Comparison of intake volume of saccharin solution before and after rotary stimulation in the four experimental groups. A:
rotary stimulation undosed; B: rotary stimulation saline dosed; C: rotary stimulation anisodamine dosed; D: control group. *P,0.05 as compared with
the before rotary stimulation respectively. The reduction in group C was significantly less than those of group A and group B in the three 24 h
intervals after rotary stimulation.
Table 3. Comparison of reduction in intake volumes (%) in
the 4 experimental groups.
group 1st 24 h2nd 24 h 3rd 24 h
A 60.0 64.967.8
B 62.1 65.868.4
C 81.1*84.6* 84.8*
D 95.4 100.4 96.8
A rotary stimulation undosed;B rotary stimulation saline dosed; C rotary
stimulation anisodamine dosed;D control group. *P,0.05 as compared with
groups A and B, respectively.
CGRP in Vestibular System of Rats with MS
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Figure 2. The CGRP positive cells in the vestibular efferent
nucleus(magnification:10620). I: triple rotary stimulation with
undosed; II: single rotary stimulation with undosed; III: single rotary
stimulation with saline dosed; IV: single rotary stimulation with
anisodamine dosed; V: control group, 4 V: 4th ventricle. Arrows show
the CGRP positive cells in the vestibular efferent nucleus.
Figure 3. The numbers of CGRP positive cells in the efferent nucleus. I: triple rotary stimulation with undoesd; II: single rotary stimulation
with undoesd; III: single rotary stimulation with saline dosed; IV: single rotary stimulation with anisodamine dosed; V: control group.*P,0.05 for
group I vs. group V and group II vs. group V; #P,0.05 for group IV vs. group II.
Figure 4. Expression of CGRPi fibers in the vestibular nuclei
(magnification:10610). I; triple rotary stimulation with undoesd; II:
single rotary stimulation with undoesd; III:single rotary stimulation with
saline dosed; IV:single rotary stimulation with anisodamine dosed; V:
control group. Arrows show the CGRPi fibers in the vestibular nuclei.
CGRP in Vestibular System of Rats with MS
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on the afferent activity arising from each vestibular receptor.
Although such an interaction is not clear in the vestibular system,
it has been reported that CGRP increases intracellular Ca2+
concentration in response to Ach in cochlear hair cells. 
CGRP has also been shown to influence the expression of nicotinic
acetylcholine receptor (nAChR) subunits in skeletal muscle which
leads to an increase in cAMP levels and acetylcholine receptor
synthesis. CGRP may have a similar regulatory role in the
vestibular system. [13,18] CGRP up-regulates synthesis of
acetylcholine receptors and alters the sensitivity of primary
afferent neurons to acetylcholine.
Recently, researchers report that the vestibular nuclei contain
numerous afferent neurons that send projections to the vestibular
efferent nucleus, some of which are CGRP cells.  This afferent
innervation provides morphological evidence that EVN receive
input from including CGRP cells.  These vestibular primary
CGRP afferent neurons may have an influence on EVN. CGRP
acts as an important co-transmitter and modulator in the afferent-
mediated activity of vestibular efferent neurons, which in turn
affect afferents in the vestibular end-organs. [13,18,24] In our
study, we observe that the level of CGRP fiber immunoreactivity
in the vestibular nucleus increases significantly in an animal model
of motion sickness, and the increase after 3 rotary stimulation
events is greater than after rotary stimulation once. It is unclear
today whether the CGRP fibers are afferent to the vestibular
efferent nucleus from CGRP afferent cells in the vestibular nucleus
or efferent from VEN to the vestibular nucleus. However, the
results of our current studies suggest that CGRP may have a
potential role in motion sickness.
In our experiments, the number of CGRPi neurons in VEN
and the level of CGRP fiber immunoreactivity in the vestibular
nucleus increased significantly in rats following rotary stimula-
tion. Our findings have provided further evidence to indicate an
important role of CGRP in the pathogenesis of motion sickness
and support additional neurophysiological and molecular biolog-
Anisodamine and scopolamine are anti-cholinergic drugs and
both are used to prevent and treat motion sickness. [25,26] It has
been proved that anisodamine and scopolamine have equal anti-
motion sickness effect. Moreover, anisodamine does not induce
drowsiness, blurred vision, or other side effects, as does scopol-
amine. Therefore, according to study of these two agents,
anisodamine is more suitable for anti-motion sickness than
scopolamine.  Accordingly, in our experiment, we selected
anisodamine as an anti-motion sickness drug and investigated the
effect of anisodamine on motion sickness and CGRP. Our results
show that anisodamine administered orally before stimulation can
alleviate the symptoms (i.e. CTA) of motion sickness (Figure1).
Importantly, this study demonstrates for the first time that this
medication can also significantly reduce the expression of CGRP
in the vestibular efferent nucleus and the vestibular nucleus of rats
that have undergone rotary stimulation (Figures 2, 5). This finding
could be very helpful not only to further support a link between
CGRP and motion sickness but also to motivate the use of CGRP
as a potential biomarker for developing a specific test or new
medication for motion sickness.
Conceived and designed the experiments: FL ZZ WX. Performed the
experiments: WX SZ XJ. Analyzed the data: ZL FL. Contributed
reagents/materials/analysis tools: ZL SZ. Wrote the paper: WX FL XJ.
Figure 5. The mean optical density of CGRP immunoreactivity in the vestibular nuclei of rats. I: triple rotary stimulation with undoesd; II:
single rotary stimulation with undoesd; III: single rotary stimulation with saline dosed; IV: single rotary stimulation with anisodamine dosed; V: control
group. *P,0.05 for group I vs. group V and group II vs. group V; #P,0.05 for group IV vs. group II.
CGRP in Vestibular System of Rats with MS
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PLOS ONE | www.plosone.org7 October 2012 | Volume 7 | Issue 10 | e47308