Temporal change in NMDA receptor signaling and GABAA receptor exypression in rat caudal vestibular nucleus during motion sickness habituation

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Temporal change in NMDA receptor signaling and GABAA receptor exypression in rat caudal vestibular nucleus during motion sickness habituation Jun-Qin Wang, Hong-Xia Li1 , Xin-Min Chen, Feng-Feng Mo, Rui-Rui Qi, Jun-Sheng Guo⁎, Yi-Ling Cai⁎ Department of Military Hygiene, Faculty of Naval Medicine, Second Military Medical University, 800 Xiang Yin Road, Shanghai, China

A R T I C LE I N FO

AB S T R A C T

Article history:

Repeated exposure to a provocative motion stimulus leads to motion sickness habituation

Accepted 21 April 2012

indicative of the existence of central processes to counteract the disturbing properties of

Available online 28 April 2012

the imposed motion. In the present study, we attempt to investigate whether NMDA and GABAA receptors in rat caudal vestibular nucleus neurons are involved in motion sickness

Keywords:

habituation induced by repeated Ferris-wheel like rotation in daily session (2 h/d). We

NMDA receptor

showed that defecation response increased and spontaneous locomotion decreased within

GABAA receptor

4 sessions (sickness phase). They recovered back to the control level after 7 sessions

Vestibular nucleus

(habituation phase). Western blot analysis found that NMDA receptor signal molecules:

Motion sickness habituation

calmodulin protein kinase II and cAMP response element-binding protein (CREB) were both

Plasticity

activated during sickness phase, while a prolonged CREB activation was also observed during habituation phase. Real-time quantitative PCR revealed an increase in c-fos and a decrease in Arc mRNA level during sickness phase. We also found an increase in GABAA receptor α1 subunit (GABAA α1) protein level in this stage. These results suggested that altered NMDA receptor signaling and GABAA receptor expression level in caudal vestibular nucleus were associated with motion sickness habituation. Furthermore, immunofluorescence and confocal laser scanning microscopy showed that the number of GABAA α1 immunolabeled neurons in caudal vestibular nucleus increased while the number of GABAA α1/Arc double labeled neurons and the average amount of Arc particle in soma of these neurons decreased during sickness phase. It suggested that GABAA receptor level might be negatively regulated by Arc protein in caudal vestibular nucleus neurons. © 2012 Elsevier B.V. All rights reserved.

⁎ Corresponding authors at: Room 809, Military Medical Building, 800 Xiang Yin Road, Shanghai, China. Fax: + 86 21 81871120. E-mail addresses: [email protected] (J.-S. Guo), [email protected] (Y.-L. Cai). Abbreviations: NMDA, N-methyl-D-aspartate; CaMKII, calmodulin protein kinase II; CREB, cAMP response element-binding; GABA, γ-aminobutyric acid; MVe, medial vestibular nucleus; SpVe, spinal vestibular nucleus; BDNF, brain-derived neurotrophic factor; LTP, long-term potentiation; CaMKIV, calmodulin protein kinase IV; MAPK, mitogen-activated protein kinase; GAPDH, glyceraldehyde phosphate dehydrogenase 1 Co-author. 0006-8993/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2012.04.041

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1.

Introduction

Motion sickness has a major influence on modern traveling activities and spaceflight. Most subjective symptoms of motion sickness are autonomic responses including pallor, nausea and vomiting, and loss of appetite. Repeated exposure to a provocative motion stimulus leads to habituation with diminution or eventual disappearance of established motion sickness reactions (Reason and Brand, 1975; Shupak and Gordon, 2006). Habituation training, as a countermeasure to motion sickness, is superior to anti-motion-sickness drugs and free of side effects (Golding and Gresty, 2005). However, the central mechanism underlying habituation of motion sickness is still unclear. Extensive evidence has confirmed that provocative head motion and altered gravitational environments can produce profound plastic alterations in the vestibular nuclei (Abe et al., 2007; du Lac et al., 1995; Kenyon and Young, 1986; Miles and Lisberger, 1981). In our previous study, we found that motion sickness habituation was associated with reduced Fos protein expression in rat caudal vestibular nucleus—the region which is comprised of portions of the medial and spinal vestibular nuclei (MVe, SpVe) (11.6–12.3 mm caudal to Bregma) located caudal to the lateral vestibular nucleus (LVe) (Cai et al., 2010; Paxinos and Watson, 2005). This area of the vestibular nucleus complex is thought to mediate autonomic responses in a variety of species during movement and change in posture (Aleksandrov et al., 1998; Miller et al., 2008; Xu et al., 2002). Expression of Fos protein in this region also underwent spatiotemporal changes during vestibular compensation following unilateral labyrinthectomy (Darlington et al., 1996; Dutia, 2010; Kim et al., 1997, 2002). These results suggested that Fos protein might be an important mediator for activity-dependent neuroplasticity in vestibulo-autonomic responses (Kaufman, 2005). Previous studies have confirmed that the transcription of the c-fos gene can be up-regulated by phosphorylation of the nuclear transcription factor cAMP response element-binding (CREB) protein at Ser-133 in response to Ca 2+ influx mediated by NMDA receptor (Flavell and Greenberg, 2008; Miyamoto, 2006). CREB was also observed to be activated in rat vestibular nucleus neurons after unilateral labyrinthectomy, suggesting the involvement in vestibular plasticity (Kim et al., 2000). In the meantime, other NMDA receptor down-stream signaling molecules including calmodulin protein kinase II (CaMKII) and brain-derived neurotrophic factor (BDNF) also play important roles in modulation of vestibular function (Bolger et al., 1999; Maingay et al., 2000; van Welie and du Lac, 2011). In the present study, in order to examine whether NMDA receptor mediated signal pathways contribute to motion sickness habituation, we have investigated the effect of repeated daily Ferris-wheel like rotation on CREB and CaMKII activity and on c-fos and BDNF transcription in rat caudal vestibular nucleus. The presence of motion sickness habituation in rats was assessed by defecation response and spontaneous locomotion, two observational indexes which can be examined without manipulation of the animals' behavior with conditioning procedures (Cai et al., 2010; Eskin and Riccio, 1966; Ossenkopp and Frisken, 1982; Ossenkopp et al., 1994; Santucci et al., 2009).

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Moreover, anatomical and electrophysiological studies have confirmed that secondary vestibular nucleus neurons also receive inhibitory inputs from the brain stem commissural pathway and the cerebellar flocculus (Epema et al., 1988; Stahl and Simpson, 1995). Extensive evidence indicates that GABAA receptor, which mediates fast inhibitory synaptic transmission, plays important roles in vestibular plasticity regulation (Chebib and Johnston, 1999; Gliddon et al., 2005; Yamanaka et al., 2000). In situ hybridization study showed that the MVe, SpVe and LVe were moderately or strongly labeled for GABAA receptor α1 subunit (GABAA α1) mRNA (Eleore et al., 2004). GABAA α1 protein was also expressed in the rat vestibular nucleus neurons (Pirker et al., 2000). Since α1 subunits are essential for the assembly of most GABAA receptors in brain (Barnard, 1998), we also investigated the temporal change in expression of this subunit in caudal vestibular nuclei neurons of rats exposed to repeated rotation stimulation. In addition, recent studies have proved that an immediate-early gene product Arc contributes to activity-

Fig. 1 – Defecation response (A) and spontaneous activity including total distance traveled (B) and rearing (C) of rats receiving 1, 4, 7, 10 or 13 daily rotation sessions or static treatment. Datum in each group was shown as mean (±S.E.). *P < 0.05, **P < 0.01 compared with corresponding Sta control group. The results showed that animals underwent sickness phase (within 4 sessions) and habituation phase (after 7 sessions) during the whole treatment process.

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dependent synaptic plasticity and adaptive behavior (Bramham et al., 2008, 2010; Rodriguez et al., 2005; Shepherd and Bear, 2011). Arc also plays an important role in synaptic homeostasis and network stability by regulating internalization of AMPA receptor (Chowdhury et al., 2006; Gao et al., 2010; Shepherd et al., 2006). However, whether Arc regulates GABAA receptor level and/or contributes to motion sickness habituation is unclear. Thus, in the present study, the temporal change in Arc mRNA level and co-expression of Arc protein with GABAA α1 in caudal vestibular nuclei neurons were also observed.

2.

Results

2.1. The effect of daily rotation on motion sickness symptoms Fig. 1 shows the changes in motion sickness symptoms during the 13 rotation sessions. A 2 (rotation condition) × 5 (treatment time: 1, 4, 7, 10, 13 days) factorial ANOVA analysis found significant effect of rotation [F (1, 20) = 41.753, P = 0.0001], time [F (4, 20)= 13.344, P = 0.0001] and rotation × time interaction [F (4, 20) = 6.677, P = 0.0001] on defecation response over the 13 rotation sessions. Fisher's Least Significant Difference (LSD) post hoc analysis found significant increase in defecation response in animals receiving 1 or 4 rotation sessions compared

with corresponding static (Sta) control animals (P< 0.01, P < 0.05) (Fig. 1A). Factorial ANOVA also revealed a significant effect of rotation [F (1, 20)= 5.237, P = 0.025], time [F (4, 20)= 33.739, P = 0.0001] and rotation × time interaction [F (4, 20) = 32.734, P = 0.0001] on total distance traveled; a significant effect of rotation [F (1, 20) =7.720, P=0.007], time [F (4, 20)=9.000, P= 0.0001], as well as rotation×time interaction [F (1, 20)=9.113, P = 0.0001] on rearing over the 13 rotation sessions. Post hoc analysis also found significant decrease in both total distance traveled and rearing in animals receiving 1 or 4 rotation sessions compared with corresponding Sta control animals (P < 0.05) (Figs. 1B, C). These results showed that animals in rotation (Rot) groups underwent sickness phase (within 4 sessions) and habituation phase (after 7 sessions) during the whole treatment process.

2.2. The effect of daily rotation on NMDA and GABAA subunits' protein level in the caudal vestibular nucleus Fig. 2 shows the changes in NMDA (NR1, NR2A/B subunit) and GABAA (α1 subunit) protein level in rat caudal vestibular nucleus during the 13 rotation sessions. Factorial ANOVA revealed a significant effect of rotation [F (1, 20) = 15.211, P = 0.001], time [F (4, 20) = 19.320, P = 0.0001] and rotation × time interaction [F (4, 20) = 6.197, P = 0.002] on GABAA α1 level over the 13 rotation sessions. Post hoc analysis found that GABAA

Fig. 2 – Western blot analysis of NR1, NR2A/B and GABAA α1 subunit in caudal vestibular nucleus of rats receiving 1, 4, 7, 10 or 13 daily rotation sessions or static treatment. (A) Representative images of western blot demonstrating NR1, NR2A/B and GABAA α1 subunit protein level in the caudal vestibular nucleus. (B) Statistical plot of data displaying the effects of daily rotation stimulation on expression of NR1, NR2A/B and GABAA α1 subunit. Values in Rot groups are expressed as percentage of their corresponding Sta control values (100%) and shown as mean (±S.E.). *P < 0.05, **P < 0.01 compared with corresponding Sta control group.

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α1 increased significantly in animals receiving 1 or 4 rotation sessions compared with corresponding Sta control animals. There was no significant effect of rotation or time on NR2 and NR2A/B level in Rot groups during the whole process (Figs. 2A, B).

2.3. The effect of daily rotation on phosphorylation of CaMKII α subunit and CREB in the caudal vestibular nucleus Fig. 3 shows the changes in phosphorylation of CaMKII α subunit (αCaMKII) and CREB in rat caudal vestibular nucleus during the 13 rotation sessions. There was a significant effect of rotation [F (1, 20) = 41.552, P = 0.0001], time [F (4, 20) = 12.161, P = 0.0001] and rotation × time interaction [F (4, 20) = 14.127, P = 0.0001] on ratio of phosphorylated αCaMKII relative to total αCaMKII protein level (p-αCaMKII/αCaMKII) over the 13 rotation sessions. LSD post hoc analysis found significant increase in p-αCaMKII/αCaMKII in animals receiving 1 or 4 rotation sessions compared with corresponding Sta control animals (P < 0.01). Meanwhile, rotation [F (1, 20) = 70.414, P = 0.0001], time [F (4, 20) = 7.518, P = 0.001] and rotation × time interaction [F (4, 20) = 6.866, P = 0.001] also had significant effect on ratio of phosphorylated CREB relative to total CREB protein level (p-CREB/CREB) over the 13 rotation sessions. Compared with corresponding Sta control animals, p-CREB/ CREB increased in animals exposed to 1, 4, 7 or 10 rotation

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sessions and reduced to baseline level after 13 sessions (P<0.01) (Figs. 3A, B).

2.4. The effect of daily rotation on c-fos, Arc and BDNF mRNA transcription in the caudal vestibular nucleus RT-PCR analysis showed that the correlation coefficient for the standard curves for GAPDH and the target molecules ranged from 0.997 to 0.999. Fig. 4 shows the changes in c-fos, Arc and BDNF mRNA level in rat caudal vestibular nucleus during the 13 rotation sessions. Factorial ANOVA analysis found significant effect of rotation [F (1, 20) = 6.242, P = 0.021], time [F (4, 20) = 3.944, P = 0.016] and rotation × time interaction [F (4, 20) = 2.734, P = 0.048] on c-fos mRNA level over the 13 rotation sessions. Compared with Sta control animals, c-fos mRNA increased in animals exposed to 1 and 4 rotation sessions (P < 0.05). The level of Arc mRNA was also affected by rotation [F (1, 20) = 10.45, P = 0.004], time [F (4, 20) = 3.493, P = 0.026] and rotation × time interaction [F (4, 20) = 3.715, P = 0.02]. LSD post hoc analysis revealed that Arc mRNA decreased in animals exposed to 1 and 4 rotation sessions compared with corresponding Sta control animals (P < 0.01, P < 0.05). No significant effect of rotation or time on BDNF mRNA level was found in Rot groups during the whole process (Fig. 4).

Fig. 3 – Western blot analysis of p-αCaMKII, αCaMKII, p-CREB and CREB in caudal vestibular nucleus of rats receiving 1, 4, 7, 10 or 13 daily rotation sessions or static treatment. (A) Representative images of western blot demonstrating p-αCaMKII, αCaMKII, p-CREB and CREB protein level in caudal vestibular nucleus. (B) Statistical plot of data displaying the effects of daily rotation stimulation on ratio of p-αCaMKII/αCaMKII and p-CREB/CREB. Values in Rot groups are expressed as percentage of their corresponding Sta control values (100%) and shown as mean (±S.E.). **P < 0.01 compared with corresponding Sta control group.

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GABAA receptor α1 subunit protein level in caudal vestibular nucleus neurons throughout the whole rotation sessions.

3.1.

Fig. 4 – Quantitative real-time PCR analysis of c-fos, Arc and BDNF mRNA level in caudal vestibular nucleus of rats receiving 1, 4, 7, 10 or 13 daily rotation sessions or static treatment. Values in Rot groups are expressed as percentage of their corresponding Sta control values (100%) and shown as mean (±S.E.). *P < 0.05, **P < 0.01 compared with corresponding Sta control group.

2.5. Co-expression of GABAA α1 subunit with Arc in the caudal vestibular nucleus neurons GABAA α1 and Arc double-labeled (GABAA α1/Arc-LI) neurons in the caudal vestibular nucleus were observed under laser scanning confocal microscopy. Arc-labeled (Arc-LI) neurons were distributed in both MVe and SpVe, while GABAA α1-labeled (GABAA α1-LI) and GABAA α1/Arc-LI neurons were mainly distributed in the SpVe. Neurons with relatively high intensity of GABAA α1 fluorescent staining generally had fewer Arc particles in the soma (Figs. 5A–F). Arc particles were mainly scattered around the nucleus of the GABAA α1-LI neurons in SpVe of animals receiving 1 or 4 rotation sessions (Figs. 4B, C), while they were relatively enriched in neuronal soma of Sta control animals and animals receiving 7, 10 or 14 rotation sessions (Figs. 4A, D–F). One-way ANOVA revealed that the amount of GABAA α1-LI and GABAA α1/Arc-LI neurons and the average amount of Arc particle per GABAA α1/Arc-LI neuron (Arc/ neuron) were significantly different between Rot and Sta control groups [F (5, 24) =35.9, P=0.0001; F (5, 24)=20.7, P=0.0001; F (5, 24)= 11.9, P=0.0001]. Dunnett-t test analysis found that the number of GABAA α1-LI neurons in the caudal vestibular nucleus was increased while the numbers of GABAA α1/Arc-LI neurons and Arc/neuron were decreased in animals receiving 1 or 4 rotation sessions and recovered to Sta control level in animals receiving more than 7 rotation sessions (Table 1).

3.

Discussion

We have shown that repeated Ferris-wheel like rotation in daily session resulted in habituation of defecation response and spontaneous locomotion in rats and induced alteration of NMDA receptor signal pathway activity and GABAA receptor α1 subunit protein level in caudal vestibular nucleus. Moreover, Arc protein was shown to be involved in regulation of

Motion sickness symptom observation

In rodents, motion sickness symptoms include pica, defecation response, as well as reductions in body temperature and spontaneous locomotion (McCaffrey, 1985; Ossenkopp and Frisken, 1982; Ossenkopp et al., 1994). Conditioned taste aversion and conditioned nausea can also be induced in rats after multiple conditioning trials pairing novel taste (conditioned stimulus) with body rotation (unconditioned stimulus) (Cordick et al., 1999; Limebeer et al., 2008). The finding that body rotation induced conditioned taste aversions are eliminated following labyrinthectomy supports the validity of a rat model of motion sickness (Ossenkopp et al., 2003). In the present study, spontaneous locomotion decreased immediately after Ferris-wheel like rotation in rats. This observation coincides with previous studies reported for rodents (Eskin and Riccio, 1966; Ossenkopp et al., 1994; Santucci et al., 2009). Meanwhile, both defecation response and spontaneous locomotion habituated to repeated rotation in daily session after about 7 rotation sessions. This time course for motion sickness habituation achievement is similar to the data previously reported for rodents and other animal species (Crampton and Locut, 1991; Mierzwinski et al., 2001; Ossenkopp and Frisken, 1982; Wilpizeski et al., 1987). However, the results of the present study are in contrast to the previous studies using pica, as a behavioral index of motion sickness in rats (McCaffrey, 1985; Morita et al., 1990). Our previous study showed that pica response habituated to repeated daily Ferris-wheel like rotation and recovered to control level after almost 31 sessions (Cai et al., 2010). It was reported that kaolin consumption can help rats recover from chemotherapy-induced illness such as anorexia and weight loss (De Jonghe et al., 2009). These evidence suggested that the ingestion of kaolin in response to stomach irritation might be related to potential therapeutic property of this non-nutritional substance (Cabezos et al., 2010; Constancio et al., 2011). Thus, it is possible that the prolonged pica response in contrast to defecation response and spontaneous locomotion after 7 rotation sessions might be a self-medicative behavior, which was also observed in other animal species (Huffman, 2003; Villalba et al., 2010), to prevent gastrointestinal malaise induced by motion stimulation in rats.

3.2.

NMDA receptor signaling

Immunohistochemistry study showed that both α and β isoforms of CaMKII were expressed in caudal vestibular nucleus neurons (Ochiishi et al., 1998). NMDA receptor subunits (NR1 and NR2A/B) were observed to be expressed in Fos-labeled neurons within the vestibular nuclei after animals were subjected to offvertical axis rotation (Chen et al., 2003a,b). Electrophysiological experiment showed that high firing rate of MVe neurons could maintain CaMKII in an activated state (Nelson et al., 2005). These results suggested that the increment of CaMKII activity in the present study might be due to the activation of NMDA receptors in caudal vestibular nucleus neurons by intense stimulation of vestibular afferent nerve during rotation stimulation. In vitro studies demonstrated that high-frequency stimulation (HFS) of

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Fig. 5 – Representative confocal microscope images showing the appearance of GABAA α1 subunit and Arc double immunostaining in the SpVe of rat in Sta control group (A) or in 1, 4, 7, 10 and 13 Rot groups (B–F). GABAA α1 labeled neurons are stained in red (TRITC labeled), while the immunoreactivity for Arc is shown as green (FITC labeled) at high magnification. Arrowheads indicate GABAA α1 single labeled neurons; double arrowheads indicate GABAA α1/Arc double labeled neurons; bars: 25 μm in every photograph.

the primary vestibular afferents can induce long-term potentiation (LTP) in the vestibular nuclei neurons through the activation of postsynaptic NMDA receptors (Capocchi et al., 1992; Grassi et al., 2009; Pettorossi et al., 2011). Since CaMKII has also been proved to be a key mediator in induction of LTP (Lisman et al., 2002), we presume that activated CaMKII by rotation stimulation might possibly induce LTP and contribute to the change of neuronal plasticity in the vestibular nucleus neurons. Our results also showed that elevated CaMKII activity

Table 1 – The number of GABAA α1 labeled (GABAA α1-LI) and GABAA α1/Arc double labeled (GABAA α1/Arc-LI) neurons and the average amount of Arc particles in soma of GABAA α1-labeled neurons in caudal vestibular   s). nucleus (x

Sta control Rot 1 d Rot 4 d Rot 7 d Rot 10 d Rot 14 d

GABAA α1-LI

GABAA α1/Arc-LI

Arc/neuron

639.20 ± 13.26 739.45 ± 12.73 ⁎⁎ 683.67 ± 18.91 ⁎ 643.61 ± 16.54 641.02 ± 12.62 639.22 ± 20.92

450.43 ± 19.39 373.35 ± 13.24 ⁎⁎ 422.56 ± 7.60 ⁎ 454.24 ± 17.77 450.27 ± 15.12 445.65 ± 16.09

14.65 ± 3.51 7.42 ± 1.14 ⁎⁎ 7.63 ± 2.30 ⁎⁎ 15.61 ± 2.96 15.87 ± 3.11 16.86 ± 4.32

⁎⁎ P < 0.0001, compared with the static control group. ⁎ P < 0.01, compared with the static control group.

was decreased to baseline level, while a persistent increase in CREB activity was still observed in habituation phase. This result is consistent with an in vitro study which showed that p-CREB still remained at high levels following an action potential burst (Fields et al., 1997). Thus, the discrepancy between CaMKII and/or CREB activity suggested that besides CaMKII, some other molecules such as calmodulin protein kinase IV (CaMKIV) and mitogen-activated protein kinase (MAPK) might also be involved in CREB activation (Miyamoto, 2006). In addition, it has been demonstrated that CREB is one of the factors that can enhance the transcription of c-fos and BDNF (Flavell and Greenberg, 2008). In the present study, we found an increase in c-fos mRNA level just in sickness phase. This observation supports the notion that p-CREB levels are not well correlated with Fos expression in vestibular nucleus and other brain regions (Chen and Herbert, 1995; Cullinan et al., 1995; Illing et al., 2002; Shiromani et al., 1995). The reason might be that c-fos transcription depends not only on p-CREB but also on other factors such as cytokines and serum response factor, and the assembly of an interdependent transcription complex directed by the concerted action of these factors is required for the regulation of c-fos transcription (Robertson et al., 1995). Meanwhile, some repressor factors, such as ATF4 which served as CREB activity inhibitor (Bartsch et al., 1995; Chen et al., 2003a,b; Kandel, 2001), might also counteract the effect of increment of p-CREB levels observed in the habituation phase. The contribution of these factors to regulation of c-fos transcription in caudal

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vestibular nucleus during motion sickness habituation awaits further investigation.

3.3.

4.

Experimental procedures

4.1.

Animals and grouping

GABAA receptor and Arc protein

In the present study, we found that GABAA α1 protein level was increased during sickness phase and recovered to control level when motion sickness habituation was achieved. Electrophysiological studies have confirmed that most commissural inhibition between the two sides of vestibular nucleus is mediated by Type II neurons releasing GABA acting on GABAA receptors on Type I neurons on the same side (Gliddon et al., 2005). During sickness phase, the bilateral Type I neurons might be activated, which was indicated by the increment of Fos expression. Thus the increment of GABAA receptor level in these neurons might undoubtedly amplify the inhibition effect of the commissural connections and serve as a compensatory response to counteract the excitatory effect induced by NMDA receptor activation. Previous studies have confirmed that Arc protein is involved in regulation of actin cytoskeletal dynamics underlying consolidation of LTP and contributes to activity-dependent synaptic plasticity and adaptive behavior such as memory storage (Bramham, 2008; Bramham et al., 2008; Rodriguez et al., 2005). The present study showed that Arc protein seemed to regulate GABAA receptor level negatively in caudal vestibular nucleus neurons. This can be supported by our immunohistochemistry experiment, which showed that (1) the number of GABAA α1/Arc-LI neurons was gradually decreased while the number of GABAA‐LI neurons was increased in the SpVe as the daily rotation session progressed; (2) neurons with more Arc particles in the soma showed relatively low intensity of GABAA α1 fluorescent staining. In the meantime, we also observed that Arc protein interacted with GABAA α1 subunit mainly in neuronal cell soma but not in dendrites where Arc mRNAs should store (Bramham et al., 2010; Dynes and Steward, 2007). An immunocytochemical study showed that GABAA α1 subunit protein expressed moderately both in dendrites and neuronal cell soma in rat MVe and SpVe (Pirker et al., 2000). It was reported that Arc mRNAs can have their subcellular distribution altered by neuronal activity (Bramham and Wells, 2007). These results suggested the possibility that Arc protein could interact with GABAA receptor in the soma. In addition, repeated rotation stimulation leads to an expansion of the distribution region for GABAA α1/Arc-LI particles in SpVe neurons, indicating the enhanced modulation effect of Arc protein on GABAA α1 level. However, the precise molecular mechanism underlying Arc/ GABAA α1 interaction has not been clarified in this study and awaits further investigation. On the other hand, the present study also showed that the temporal change of CREB activity is in discrepancy with that of Arc expression level in caudal vestibular nucleus. Recent work has identified other response elements including serum response elements, myocyte enhancer factor-2 protein and the synaptic activity-responsive element in the Arc promoter underlying its activity-dependent transcriptional regulation (Kawashima et al., 2009; Pintchovski et al., 2009). Further studies will be undertaken to examine whether transcription factors acting on these elements are involved in regulation of Arc expression in caudal vestibular nucleus during motion sickness habituation.

Adult male Sprague–Dawley (SD) rats weighing 250–300 g were used. All animal protocols and procedures described were approved by the Animal Use and Care Committee for Research and Education of the Second Military Medical University (Shanghai, PR China). The animals were housed under a 12-h light:12-h dark cycle (temperature: 22 ± 2 °C and lighting: 8:00–20:00) with free access to food and water. Efforts were made to minimize the number of animals used and their suffering. Eighty animals were used for western blot and realtime quantitative PCR (RT-PCR) test and randomly divided into five rotation treatment (Rot) groups receiving 1, 4, 7, 10 or 13 rotation sessions on a daily basis (2 h/d), respectively, and five corresponding static control (Sta) groups (kept in the restrainer near the rotation device when Rot animals were being rotated) (n = 8 in each group). Another thirty animals were used for immunohistochemistry study and divided into five Rot groups treated the same as above and one Sta group (kept in the restrainer for 2 h but not rotated) (n = 5 in each group). 4.2.

Rotation device and procedures

The rotation device and detailed rotation methods were as described previously (Cai et al., 2007). In brief, plexiglass containers suspended on a metal frame revolved about an axis parallel to the floor. It started to rotate in clockwise direction at a constant angular acceleration of 16°/s2. After the angular velocity reached 120°/s, it began to slow down at a constant angular deceleration of 48°/s2. After 1 s of pause, the container continued to rotate in counter-clockwise direction in the same manner as above. The magnitude of angular velocity and centrifugal acceleration ranged from 0 to 120°/s to 0 to 2.22 m/s2, respectively. A clockwise-pause-counterclockwise cycle lasted about 21 s. All rats were stimulated for 2 h consecutively in this manner in complete darkness with temperature maintained at 22 °C according to the preliminary studies. In addition, all animals were pre-adapted to the treatment equipment for 2 h per day for 3 days prior to the beginning of all experiments. The adaptation and rotation stimulation procedures were performed during 6:00–8:00 p.m. with the temperature maintained at 22 °C. 4.3.

Motion sickness symptom observation

Immediately after each treatment session, animals used for western blot and RT-PCR test were taken out of the plexiglass containers of the rotation device and were tested for spontaneous locomotion. The number of fecal granules deposited by each animal in the plexiglas restrainer was counted (Cai et al., 2010). In spontaneous locomotion test, locomotion (total distance traveled) and exploratory activity (rearing) were measured by an animal behavior test system (JLBehv-LR4, jlsofttech, Shanghai, China). The apparatus consisted of a dark 40× 40× 45 cm rectangular chamber with the floor marked with a 16× 16 grid. Testing was conducted in a soundproof room. The animal

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was placed in the center of the chamber and left undisturbed for 3 min. The behavior and the locomotion tracking of the animals were recorded by an infrared digital video camera. Total distance traveled and the number of rearing (standing on hind legs with the body inclined vertically, forequarters raised) during the 3 min performance were measured by a commercial software (DigBehv 2.0, jlsofttech, Shanghai, China). 4.4.

Tissue preparation

In western blot and RT-PCR experiments, animals in each group were anesthetized with an overdose of sodium pentobarbital (100 mg/kg, i.p.) and perfused transcardially with 60 ml chilled saline. The brains were immediately dissected out and cooled in iced saline for 1 min. A brainstem block including the vestibular nucleus complex was made in order to dissect caudal portion of the vestibular nucleus. Coronal sections were cut from the brainstem block at every 200 μm in a cryostat, and each thick section (200 μm) was mounted onto a slide glass. Approximately 4 thick sections were made from a single brainstem block. The medial vestibular nucleus (MVe) and the spinal vestibular nucleus (SpVe) located between Bregma − 11.6 mm and −12.3 mm according to rat brain atlas of Paxinos and Watson (2005) were dissected with blunted 22 gauge needles. Dissected tissues from bilateral side of thick sections in one animal were pooled as one sample, frozen on dry ice and stored at −80 °C. Half sample from each animal was used for western blot and another half was used for RTPCR test. The remaining parts of brainstem thick sections were further fixed with 4% paraformaldehyde at 4 °C for 1 h; they were then placed in 0.1 mol/l PB containing 30% sucrose overnight at 4 °C before made into 20-μm thick sections. One out of 5 sections was selected for Nissl-staining to verify the precise location of the dissected tissues. In immunohistochemistry experiment, animals were anesthetized with an overdose of sodium pentobarbital (100 mg/kg, i.p.) and then perfused transcardially with 100 ml chilled saline, followed by perfusion with 500 ml 0.1 mol/l phosphate buffer (PB, pH 7.4) containing 4% paraformaldehyde. The brains were removed, postfixed with 4% paraformaldehyde at 4 °C for 1 h; a brainstem block including the caudal portion of the vestibular nucleus was made and placed in 0.1 mol/l PB containing 30% sucrose overnight at 4 °C before made into 20 μm-thick sections. One out of every 3 consecutive sections (11.6 mm–12.3 mm caudal to Bregma) was selected for immunostaining. 4.5.

Western blot analysis

The tissue sample was abraded and lysed in 1 ml tissue and cell lysis solution plus protease and phosphatase inhibitors (Formantas), then centrifuged at 10,000 g for 5 min at 4 °C. The protein content of the supernatant was determined spectrophotometrically using the bicinchoninic acid method. Equal amount of protein (50 μg per lane) from each sample was loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), electrophoresed, and transferred onto nitrocellulose membrane (Millipore Corp, Bedford, MA, USA) using a Mini-Trans-Blot Electrophoresis Transfer Cell (Bio-Rad, Hercules, CA, USA). Nonspecific binding of antibodies was prevented by incubating membranes in 3% bovine serum albumin (BSA) in

37

Tris-buffered saline containing Tris-buffered-saline-Tween 20 (TBST: 10 mM Tris–HCl, 150 mM NaCl, 0.05% Tween-20). The membranes were incubated overnight at 4 °C with the following primary antibodies: anti-NR1 (1:1000; Abcom Ltd, Hong Kong); anti-NR2A/B (1:1000; Abcom Ltd, Hong Kong); anti-GABAA α1 (1:1000; Abcom Ltd, Hong Kong); anti-p-αCaMKII (1:1000; Abcom Ltd, Hong Kong); anti-αCaMKII (1:1000; Abcom Ltd, Hong Kong); anti-p-CREB (1:1000; Millipore Crop, Bedford, MA, USA) or anti-CREB (1:1000; Millipore Crop, Bedford, MA, USA) in TBST with 1% BSA. After being extensively washed with TBST, the membranes were incubated for 2 h, at room temperature, with peroxidase-labeled secondary antibodies (anti-rabbit IgG, Jackson, West Groove, PA) at 1:5000 dilution. The bands were then visualized by an ECL system (Millipore Corp, Bedford, MA, USA). The density of the bands was analyzed by the Gel Doc image system (BioRad, San Diego, CA, USA). Signal intensities were normalized against the internal control (β-actin). The phosphorylation levels for αCaMKII and CREB were calculated relative to αCaMKII (p-αCaMKII/αCaMKII) and CREB (p-CREB/CREB) total protein levels. 4.6.

Real-time quantitative PCR

Tissue samples were homogenized in Trizol reagent (Invitrogen, USA) and total RNA was purified. First strand complementary DNA (cDNA) was synthesized by retrotranscription using oligodT primers and PrimeScript RT Reagent Kit (Takara, Japan) according to the manufacturer's instructions. Real-time PCR reactions were carried out in a Rotor-Gene (RG-3000A, Corbett Research) PCR machine and the detailed procedures were as follows: an initial denaturation step at 95 °C for 5 s, followed by 40 cycles of a 95 °C denaturation for 5 s, 60 °C annealing for 20 s, and 72 °C extension for 30 s. The amounts of cDNA per sample were determined using a SYBR Premix Ex Taq™ kit (Takara). Progression of PCR reaction was assessed by changes of the SYBR Green dye fluorescence attached to double stranded DNA. All values were normalized to the housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH). The primers used for real-time PCR were: Fos: sense primer 5′GCATCGGCAGAAGGGGCAAAGT-3′; Fos: antisense primer 5′-TGATCTGTCTCCGCTTGGAGCGT-3′; Arc: sense primer 5′-TGCATCAGTGTCCAGGGCCCTT-3′; Arc: antisense primer 5′-ATGAATCACTGCTGGGGGCCAG-3′; BDNF: sense primer 5′TCAGAATGAGGGCGTTTGCGT-3′; BDNF: antisense primer 5′TCACCTGGTGGAACATTGTGGC-3′; GAPDH: sense primer 5′GGCTCTCTGCTCCTCCCTGTTCTA-3′; GAPDH: antisense primer 5′-CGTCCGATACGGCCAAATCCGT-3′. 4.7.

Immunohistochemistry

The selected sections were washed in 0.01 M phosphatebuffered solution (PBS) (PH; 7.4) and incubated in a rabbit anti-GABAA α1 IgG (1:1000; Abcom Ltd, Hong Kong) and goat anti-Arc IgG (Santa Cruz Biotechnology Inc.; 1:1000) for 24 h at 4 °C. After washing in PBS, the sections were incubated in TRITC-labeled donkey anti-rabbit IgG (1:200; Jackson, West Groove, PA) for 4 h at room temperature. After washing, the sections were further incubated in FITC-labeled rabbit antigoat IgG (1:200; Jackson, West Groove, PA) for another 4 h at room temperature and washed in PBS. Then the sections

38

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were mounted onto a gelatin-coated glass slide for observation under a fluorescence microscope (BX-60; Olympus) with appropriate filters for FITC (excitation wavelength: 490 nm, emission wavelength: 520 nm), and TRITC (excitation wavelength: 543 nm, emission wavelength: 570 nm). Then, the sections exhibiting positive staining were examined under a laser confocal scanning microscope (Confoco ST5, Leica, Germany) by using laser beams 570 nm for TRITC, 488 nm for FITC. Images from each channel of the same section were superimposed and the photographs were taken with a digital camera. Sections for each rat from comparable rostrocaudal levels of the vestibular nucleus (10 sections per animal) were chosen for cell counting. The numbers of GABAA α1, GABAA α1-LI and GABAA α1/Arc-LI neurons were counted in bilateral MVe and SpVe located between Bregma −11.6 mm and −12.3 mm coronal section. In every GABAA α1/Arc-LI neuron, the number of Arc particle in soma was also counted and the average amount of Arc particle per neuron (Arc/neuron) was calculated. 4.8.

Statistical analysis

Statistical analysis was performed using the SPSS v13.0 statistical program. Two-factorial analysis of variance (ANOVA) was performed using General Linear Protocol to examine whether there was any significant effect of rotation, time and/or interaction effect of rotation × time throughout the whole treatment process. Fisher's LSD post hoc test was used to analyze the difference between Rot and corresponding Sta group, when a significant rotation × time interaction effect was obtained. In western blot and RT-PCR experiments, all results in Rot groups are expressed as a normalized percentage of their corresponding Sta control groups. In immunohistochemistry experiment, one-way ANOVA combined with Dunnett-t test was performed to analyze the number of GABAA α1-LI, GABAA α1/Arc-LI neurons and Arc/neuron in the caudal vestibular nuclei (MVe and SpVe). All data presented were expressed as mean ± SEM. Statistical significance was judged at P < 0.05.

Acknowledgments We would like to thank Prof. Jian Lu (Second Military Medical University) for his assistance in statistical analysis. This study is supported by a grant from the National Natural Foundation of China (30800544).

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