Download Immunohistochemical Identification of Phosphorylated Extracellular

대한평형의학회지 제 2 권 2 호 2003 ; 170-174
Immunohistochemical
Identification
of
Phosphorylated
Extracellular Signal-Regulated Kinase1/2 in Rat Vestibular
Nuclei by Unilateral Labyrinthectomy
원광대학교 의과대학 생리학교실
최명애, 최동옥, 김광영, 이문영, 박병림, 김민선
Immunohistochemical Identification of Phosphorylated Extracellular SignalRegulated Kinase1/2 in Rat Vestibular Nuclei by Unilateral Labyrinthectomy
Myoung Ae Choi, Dong Ok Choi, Kwang Yong Kim, Moon Young Lee, Byung Rim Park,
Min Sun Kim
Department of Physiology, Wonkwang University School of Medicine and Vestibulocochlear Research Center at Wonkwang
University Iksan 570-749, South Korea
This study evaluated the expression of phosphorylated signal-regulated kinase1/2 (pERK1/2), which is one of the
main factors regulating transcription of the cfos oncogene in neurons, in the vestibular nuclei of Sprague-Dawley rats
following unilateral labyrinthectomy (UL). Surgical UL was performed to eliminate afferent signals from the peripheral
vestibular receptors in the inner ear, under a surgical microscope, 2 hours after anesthesia. Significant numbers of
pERK1/2 immunoreactive neurons were seen in the superior, medial, and inferior vestibular nuclei. There were more
pERK1/2 immunoreactive cells in the vestibular nuclei contralateral than in the vestibular nuclei ipsilateral to the
injured labyrinth, which resulted in significant asymmetric expression of pERK1/2 immunoreactive cells. Subsequently,
the pERK1/2 immunoreactivity decreased rapidly, disappearing 90 min after labyrinthectomy. No pERK1/2 labeling was
seen in the lateral vestibular nucleus. These results suggest that intracellular signal pathways for the activation of
extracellular signal-regulated kinase in the vestibular nuclei are involved in lesion-neural plasticity in the vestibular
system
Key Words: Labyrinthectomy, pERK1/2, Vestibular Nucleus, Vestibular compensation
Introduction
Unilateral labyrinthectomy (UL) produces characteristic ocular and postural deficits, as well as autonomic
∙교신저자 : Min Sun Kim
Department of Physiology, Wonkwang University
School of Medicine, Iksan 570-749
Tel: 82-63-850-6779, Fax: 82-63-852-6108,
E-mail: [email protected]
170
symptoms. However, recovery from most of these
symptoms occurs within a few days, so-called vestibular
compensation (VC). VC is one of experimental models
for lesion-induced neural plasticity in CNS. There is
large body of results that show asymmetric spatiotemporal changes in the expression of several inducible
transcriptional factors in the vestibular nuclei 1,2,8,10). In
addition, the phosphorylated form of the cAMP/calcium
response element binding protein (pCREB), one of the
Myoung Ae Choi, et al
constitutional transcription factors is detected in the
12)
vestibular nuclei within 12 hours following UL . Other
phosphorylated proteins are also detected in the medial
18)
vestibular nucleus in vitro following UL .
Extracellular regulated protein kinase 1/2 (ERK1/2),
one family of mitogen activated protein kinases, is
involved in a complex intracellular signaling cascade that
controls various neurobiological effects, including
neuronal differentiation and synaptic plasticity, producing
activity-dependent regulation of neuronal function in the
4,15,20)
. ERK1/2 is activated via
nervous system
phosphorylation catalyzed by MAPK/ERK kinase in
17,21)
response to neuron excitatory stimuli .. The
phosphorylated form of ERK1/2 protein (pERK1/2), an
active form, is also crucial for controlling the
transcription of inducible transcription factors via the
17)
phosphorylation of CREB or ELK-1 .
Little is known about the change in the expression of
pERK1/2 in the vestibular nuclei following unilateral
injury of the vestibular end-organs. Therefore, this study
evaluated the spatiotemporal changes in pERK1/2
expression in the VN following UL.
Experimental Methods and Materials
Sprague-Dawley rats, weighing 250-300 g, were
divided into two groups: a control group (CON) with an
intact labyrinth and a surgical unilateral labyrinthectomy
(UL) group. The procedures used were approved by the
Institutional Ethics Committee on the Experimental Use
of Animals. Surgical UL was performed, as described in
detail previously [11]. Briefly, after chloral hydrate
anesthesia (300 mg/kg, i.p.), the ossicular bones in the
left middle ear cavity were removed to open the oval
window through a ventral approach under a surgical
microscope. The membrane labyrinth was destroyed
surgically with a small right-angled hook and aspirated
with a suction pump through a small opening in the oval
window. UL was confirmed by the appearance of eye
deviation just after UL under anesthesia.
After UL, animals were sacrificed at 5 (n=6), 30
(n=6), or 120 min (n=6) for immunohistochemical
procedures. The procedures used for the immunohistochemistry and image analyses have been described
9)
in detail previously . Briefly, under deep anesthesia (1.5
g/kg, i.p.), the rats were perfused transcardially with
0.9% saline, and fixed with 4% paraformaldehyde
dissolved in phosphate-buffered saline (pH 7.4). The
sucrose-embedded brain stem was sectioned at 40-μm
thicknesses on a cryostat. Non-specific binding sites were
blocked with normal goat serum (1:50) for 30 min at
room temperature. Primary anti-rabbit polyclonal
pERK1/2 antibody (1:1000)(Cell Signaling Technology,
MA, USA) was applied overnight at 4℃. The following
day, the tissue sections were incubated with secondary
antibody and then with avidin-biotin complex for 1 h at
room temperature. The bound complex was visualized by
incubating the tissue with 0.05% diaminobenzidine and
0.003% hydrogen peroxide. For quantification, pERK1/2
positive neurons were counted using a digital image
analysis system (Image-Pro, MD, USA) in the VN at 4
16)
different levels from rostral to caudal . All the data are
presented as the mean±S.D. The statistical significance
of differences was assessed using SPSS (SPSS Inc., IL,
USA). Values with P<0.05 were considered significant.
Results
A few pERK1/2-like immunoreactive (LI) neurons
were seen in the medial and inferior vestibular nuclei,
but none in the superior and lateral vestibular nuclei in
the control group (a sham operation without UL). There
was constitutional expression of pERK1/2-LI neurons in
the solitary nuclei and some reticular nuclei of the brain
stem (n=3)(data not shown). As compared with the
control, there was significant expression of pERK1/2-LI
neurons in the superior, medial, and inferior vestibular
nuclei bilaterally, 5 min after UL. In most of the
pERK1/2-LI neurons, pERK1/2 protein appeared in the
cytoplasm of the cell body and the axonodendrite
processes. In some of the pERK1/2-LI neurons, pERK1/2
protein appeared in the nucleus. However, this induction
171
ERK activation of vestibular nuclei by labyrinthectom
of pERK1/2-LI neurons was asymmetric in that there
were many more such pERK1/2-LI neurons in the
contralateral vestibular nuclei than in the vestibular
nuclei ipsilateral to the lesion (p<0.01)(Fig. 1, 2). The
expression of pERK1/2-LI neurons in the VN peaked
within 5 min after UL, decreased rapidly by 30 min, and
finally disappeared by 90min after UL. The decrease
between 15 and 30 min was significant, and there was a
subsequent continuous decline to very low values at 90
min (Fig. 3). By contrast, UL caused no immunoreactivity for pERK1/2 in the lateral VN (LVN).
Discussion
Fig. 1. Photomicrographs depicting the expression of perk
1/2 immunoreactivity in the medial vestibular nucleus
(MVN) of control and unilateral labyrinthectomized (UL)
rats. In UL rats animal was sacrificed, perfused for
immunohistochemical staining 5 min after injury. CONT,
contralateral side; IPSI, ipsilateral side; PrH, prepositus
hypoglossi. Sold bar indicates 200mm.
In this study, UL resulted in very rapid activation and
deactivation of ERK1/2 protein in the VN bilaterally,
except the lateral vestibular nucleus. Moreover, the
activation of ERK1/2 was greater in the VN contralateral
to the injured side 5 min after UL. This asymmetric
regional distribution of pERK1/2 in the VNC 5 min after
SV N
–10.5m m
LVN
MVN
IV N
–11.3m m
–11.6m m
15 cells
CONT
IP SI
B regm a –12.0m m
Fig. 2. Bar histograph showing number of pERK immunoreactive neurons in the vestibular nuclei at 4 different levels
5 min after UL. Values are mean±S.D.. Number of rats analyzed is 6. SVN, superior vestibular nucleus; LVN, lateral
vestibular nucleus; MVN, medial vestibular nucleus; IVN, inferior vestibular nucleus. Values are mean±S.D.
172
Myoung Ae Choi, et al
**
Number of pERK1/2 (+) Cells
60
CO NT
IPSI
50
40
30
20
*
10
0
0
5
30
90
Post-U L Tim e (m in)
Fig. 3. Temporal changes of pERK1/2 immunoreactive neurons in the ipsilateral
(IPSI) and contralateral (CONT) medial vestibular at level of 11.6 mm from the
bregma following UL. "0" in X-axis indicates sham operation in control group.
Significant difference between CONT and IPSI : *P<0.05; **P<0.01.
UL is very similar to that of some transcriptional factors,
such as pCREB and cFos proteins, induced by UL1,10,12).
Deafferented vestibular nuclear neurons are essentially
silent, whereas vestibular nuclei neurons on the
unlesioned side have normal or increased resting
discharges immediately after UL23). Therefore, part of the
rapid asymmetric phosphorylation of ERK1/2 protein in
the vestibular nuclei just after UL may be attributed to
asymmetry of the electrical activity between the
vestibular nuclei bilaterally.
Elevated intracellular calcium levels resulting from the
activation of excitatory glutamate receptors are an
essential mediator of the activation of ERK postsynaptic
ERK signaling in CNS neurons4). Glutamate is one of the
major excitatory neurotransmitters required for the
transmission of excitatory afferent signals from peripheral
vestibular receptors to the central vestibular neurons, as
well as for excitatory synaptic input from the vestibular
commissural fibers5,14,22,25). Stimulation of the ipsilateral
vestibular afferent nerve and commissural fibers increases
the intracellular calcium level in MVN neurons, but not
after treatment with an NMDA receptors antagonist25).
An in vivo microdialysis study7) showed that the
glutamate concentration in the ipsi-lesional MVN
gradually decreased over 4 hours, whereas it increased in
the contra-lesional MVN immediately after UL.
Ipsilateral intra-VNC injections of a noncompetitive
NMDA receptor antagonist, MK-801, immediately before
UL, transiently reduced the frequency of SN, indicating
that NMDA receptor channels in the ipsilateral VNC are
active at the time of UL19).
One of the well-known functions of the ERK/MAPK
intracellular cascade in CNS neurons is the involvement
of different kinds of activity-dependent neural plasticity,
including roles in long-term potentiation, and conditioned
taste aversion4,6). Several lines of evidence have shown
that UL results in the expression of several transcription
factors in the vestibular nuclei and the formation of
NMDA-mediated neural plasticity at cellular or
behavioral levels at the early stage of vestibular
compensation3,13,24). Combined with previous results, the
ERK/MAPK intracellular cascade appears to play an
important role in the initiation of lesion-induced
plasticity in the vestibular system of rats.
Acknowledgements: This study was supported by a
grant of Vestibulocochlear Research Center from the
Ministry of Science and Engineering, Republic of Korea
173
ERK activation of vestibular nuclei by labyrinthectom
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