Effect of MK801 on cFos-like protein expression in the medial vestibular nucleus at early stage of vestibular compensation in uvulonodullectomized rats

Neuroscience Letters 231 (1997) 147–150

Effect of MK801 on cFos-like protein expression in the medial vestibular nucleus at early stage of vestibular compensation in uvulonodullectomized rats Min S. Kim, Byung K. Jin, Sang W. Chun, Moon Y. Lee, Sung H. Lee, Jae H. Kim, Byung R. Park* Department of Physiology, Wonkwang University School of Medicine and Medicinal Resources Research Center of Wonkwang University, Iksan, 570-749, South Korea Received 7 July 1997; accepted 16 July 1997

Abstract The purpose of this study was to evaluate the effect of uvulonodullectomy (UNL) on the expression of cFos-like protein (FLP) in the medial vestibular nucleus (MVe) during vestibular compensation and effect of MK801, an N-methyl-d-aspartate (NMDA) antagonist, on FLP expression in the brain stem nuclei at 6 h after unilateral labyrinthectomy (ULX) with UNL in Sprague–Dawley rats. Immunohistochemical staining was performed to visualize FLP in the brain stem nuclei and FLP-positive cells were counted by image analyzer. Lesion-induced asymmetric expression of FLP in the bilateral MVe was observed and maintained up to for 72 h in the ULX group, and 120 h in the UNL + ULX group. Moreover, spatial pattern of FLP expression in the bilateral MVe exhibited the marked difference between the ULX and UNL + ULX groups. MK801 treatment 6 h after ULX showed significant increase in the number of FLP in contralateral MVe (cMVe) of the ULX group, but decrease in cMVe of the UNL + ULX group. These results suggest that the lesion of vestibulocerebellum delays the temporal recovery of FLP expression in MVe and the vestibulocerebellar NMDA receptors relate to FLP expression in MVe.  1997 Elsevier Science Ireland Ltd. Keywords: Unilateral labyrinthectomy; Uvulonodular lobes; MK801; cFos protein; Vestibular compensation

Asymmetries of vestibulo-ocular and vestibulospinal reflexes are the most salient features occurring following unilateral labyrinthectomy (ULX). Some of these asymmetries gradually disappear with a time, known as vestibular compensation (VC), which might be achieved by rebalance of neuronal activity in the central vestibular system through lesion-induced central nervous system (CNS) plasticity [3,23]. It has been suggested that N-methyl-d-aspartate (NMDA) receptor, a subtype of glutamate receptors, may take part in an important role for induction of VC [6,24,26]. Recently, many studies have demonstrated that systemic or intraventricular injection of NMDA receptor antagonists could induce vestibular decompensation at an early stage of VC [4,13,21,22,25]. cFos protein, one of the immediate early gene products, can be expressed by a variety of stimuli in neurons of CNS, considered as a useful marker for detect* Corresponding author. Tel.: +82 653 506773; fax: +82 653 526108; e-mail: [email protected]

ing changes in neuronal activity [18]. Recent studies have shown the induction of cFos expression in the vestibular nuclei in response to otolith stimulation, ULX, or cerebellar flocculectomy [1,5,10–12,14,16,19,20]. We have previously reported that cerebellar uvulonodullectomy (UNL) delays vestibular compensation and does not produce vestibular decompensation but behavioral improvement following systemic injection of MK801, a channel blocker of NMDA receptor, 6 h after ULX in rats [13]. The present study determined the MK801 effects on temporal and spatial changes in cFos expression in the brain stem nuclei, especially medial vestibular nuclei (MVe) during VC in rats with UNL. Sprague–Dawley rats weighing 250–300 g were used and divided into two groups: only ULX (n = 35) and UNL preceding ULX (UNL + ULX; n = 35). ULX and UNL were described previously in detail [13]. Briefly, all animals were anesthetized with chloral hydrate (100 mg/kg, i.p.) and received surgical left labyrinthectomy through ventral

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approach under a surgical microscope. Before ULX the cerebellar uvular and nodular lobes were ablated by gentle suction with an aspiration pump under a surgical microscope. Animals received intraperitoneally a single injection of MK801 (RBI Co.) dissolved in sterile saline (1.0 mg/ml) with dosage of 1.0 mg/kg or saline as a control 6 h after ULX, and were sacrificed 2 h after drug treatment. cFos protein expression was detected by immunohistochemistry as detailed elsewhere with some modifications [9]. Briefly, all animals were deeply anesthetized with chloral hydrate, transcardially perfused, fixed in 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (PB) and decapitated. Then brains were removed, post-fixed, rinsed in PB, and immersed in 30% sucrose solution for 1–2 days at 4°C. The tissue was sectioned with a thickness of 40 mm on a freezing microtome, incubated for 30 min with 6% hydrogen peroxide (H2O2), rinsed two times for 10 min with 0.1 M phosphate buffered saline (PBS) containing 5% dry milk, incubated with 0.8% Triton X-100 dissolved in 0.1 M PBS containing 0.5% bovine serum albumin (PBS–BSA). After a brief wash, tissue was incubated overnight at room temperature with cFos polyclonal antibody (Ab-2; Oncogene; diluted 1:250). The following day tissue was rinsed with PBS–BSA, incubated with a biotinylated secondary antibody (goat anti-rabbit; DAKO) and ABC Elite Kit (Vector Lab.). The neurons with Fos-like immunoreactive (FLI) nuclei were visualized by incubating tissue with 0.05% diaminobenzidine HCl (DAB) and 0.003% H2O2. After DAB reaction the tissue was rinsed with 0.1 M PB, mounted on gel coated slides, air-dried, dehydrated, coverslipped with Permount (Fisherchemical), and analyzed by brightfield microscopy. For quantification, only FLI neurons in MVe

were counted using a digital image analysis system(Image Pro Plus, USA). All data are represented as the mean ± SE. In rats without ULX as a control, few of the FLI neurons appeared in some brain stem nuclei including MVe and inferior olivary nuclei (IO) (data not shown). However, single treatment of MK801, but neither saline injection nor UNL increased FLI labeling neurons in these nuclei (data not shown). Compared to control, ULX produced marked induction of FLI neurons in the bilateral MVe, prepositus hypoglossi, and beta nuclei of IO 2 h after ULX. This induction was asymmetrical in that the number of FLI neurons contralateral MVe (cMVe) were much higher than ipsilateral MVe (iMVe) to the lesioned side (P , 0.05) (Figs. 1a and 3a). Six hours after ULX, however, there was a significant reduction of FLI neurons in cMVe than that in iMVe so that the number of FLI neurons was slightly higher in iMVe than cMVe (Figs. 1b and 3a). Asymmetric FLI labeling between iMVe and cMVe was also observed up to 24 h after ULX (P , 0.05) and disappeared 72 h after ULX. The number of FLI neurons in the bilateral MVe returned to control 120 h after ULX (Fig. 3a). In the UNL + ULX group, spatial pattern of cFos-like protein (FLP) expression in MVe was similar to that of the ULX group except a difference in the number of FLI neurons 2 h after ULX (P , 0.05) (Figs. 1c and 3b). The number of FLI neurons of cMVe was, in marked contrast with the ULX group, significantly higher than that of iMVe 6 and 24 h after ULX (P , 0.05) (Figs. 1d and 3b). Furthermore, bilateral MVe continuously exhibited c-Fos expression 72 h after ULX. FLI labeling returned to basal level of control in cMVe, but remained higher than that of control in iMVe 120 h after ULX (Fig. 3b). The ULX group with treatment

Fig. 1. Immunohistochemical staining of cFos-like protein (FLP) expression in the bilateral medial vestibular nuclei (MVe) and prepositus hypoglossi (PrH) 2 and 6 h after either unilateral labyrinthectomy (ULX) or cerebellar uvulonodullectomy (UNL) + ULX. Following lesion, animals were anesthetized, perfused and fixed with 4% paraformaldehyde. Brains were removed, immersed in 30% sucrose, sectioned with a thickness of 40 mm, and incubated with cFos antibody. After subsequent reactions, FLP expression was visualized under bright microscope. Note the significant induction of FLP expression in the bilateral MVe caused by ULX and changes in number and position of FLP expression during vestibular compensation 2 and 6 h after either ULX (a,b) or UNL + ULX (c,d). IPSI, ipsilateral to ULX; CONTRA, contralateral to ULX. Scale bar, 400 mm.

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Fig. 2. MK801 effects on FLP expression in the bilateral MVe, and PrH 6 h after single treatment of either MK801 or saline (SAL) (as a control) in ULX (a,b) or UNL + ULX-lesioned rats (c,d), respectively. Animals were sacrificed 2 h after intraperitoneal injection of MK801 or SAL for cFos immunohistochemistry. Other notations are the same as Fig. 1. Note the marked reduction of FLP expression in the bilateral MVe in UNL + ULX-lesioned rats following MK801 treatment.

of MK801 produced a significant increase in the number of FLI neurons in cMVe (P , 0.05), but a slight decrease in iMVe compared to those of the ULX group receiving saline 6 h after ULX (Figs. 2a,b and 3c). Injection of MK801 significantly reduced FLP expression in the bilateral MVe, especially in cMVe of the UNL + ULX group (P , 0.5) (Figs. 2d and 3d). Although the marked induction of FLP expression in cMVe at 2 h after ULX is controversial [14,15], consistency with observations reported by others was the appearance of FLP expression in the bilateral MVe immediately after ULX and time-dependent change in cFos expression during the process of VC [1,5]. These discrepancies may be, in part, be attributed to species difference or experimental methods used for each study. Considering that the induction of cFos protein expression in neurons occurred within 20 min by transsynaptic depolarization [18] a lot of FLI neurons in the bilateral MVe 2 h after ULX may result from transsynaptic excitation of CNS connecting with vestibular nuclei. Because ablation of vestibular end organ removes primary vestibular afferent activity, thus resulting in an imbalance between the bilateral MVe [3,23] it might be considered that this imbalance is a possible cue to increase more FLI neurons in cMVe than in iMVe 2 h after ULX. One of the noteworthy findings in this study was a sudden reduction in the number of FLI neurons in cMVe observed in the ULX group, but not in the UNL + ULX group 6 and 24 h after ULX. Recently, Kitahara et al. [14] reported that unilateral or bilateral flocculectomy within 7 days after ULX produces either expression of FLI neurons in cMVe or vestibular decompensation. It has been known that high frequency electrical stimulation to primary vestibular afferent nerve brought out NMDA receptor-mediated long term

depression (LTD) of field potentials in MVe [7]. Therefore, one of the possible mechanisms accounting for a sudden decline of FLI in cMVe may be the decreased cMVe neuronal excitability by inhibitory synaptic input from vestibu-

Fig. 3. Quantification of FLP positive neurons during vestibular compensation (a,b) and 2 h after MK801 or saline treatment (8 h after ULX) (c,d) in the bilateral MVe. Using image analyzer (Image Pro Plus, USA), the number of FLP (+) neurons were counted from digitized image captured by monochrome CCD camera mounted on microscope. Six rats were used for each group and values are expressed as the mean ± SE. Unpaired t-test was used for statistical analysis and significant difference was defined as P , 0.05 for all analysis. *P , 0.05; significant difference between IPSI and CONTRA (a,b) or between saline and MK801 treatment (c,d).

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locerebellum and LTD within cMVe itself. Several lines of evidence have demonstrated that the vestibulocerebellar lesion delayed loss of spontaneous nystagmus and recovery of gain, phase of vestibulo-ocular reflex at an early stage of VC [2,8,9,13,14] and increased resting activity of cMVe within 2 days after ULX in cat [17]. Furthermore, in the present study, the UNL + ULX group showed the delayed recovery of FLP expression in MVe during VC. Taken altogether, these results strongly suggest that vestibulocerebellum may be involved in the initiation of VC by altering electrical and molecular events in MVe after ULX. We have previously demonstrated that treatment with MK801 produces vestibular decompensation in the ULX group, but accelerates vestibular compensation in the UNL + ULX group [13]. In the present study, the striking finding was that treatment with MK801 had different effects on FLP expression in MVe in two experimental groups (ULX vs. UNL + ULX). These results can support the hypothesis that MK801-induced vestibular decompensation may result from the increased cMVe activity caused by disinhibition of inhibitory NMDA receptor systems [15], but MK801induced acceleration of vestibular compensation is due to the decreased cMVe activity by inhibition of excitatory NMDA receptor systems. In summary, results presented here strongly suggest that inhibitory signals from the vestibulocerebellum and inhibitory NMDA receptor systems related to central vestibular system may play a pivotal role for the lesion-induced plasticity in vestibular system of rats.

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