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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. 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