| Motion sickness (MS) refers to the normal universal physiological response tounusual perception of motion, whether real or apparent, and remains an important problemin modern traveling activities and virtual reality environments. Prolonged exposure to aprovocative motion stimulus leads to diminution and eventual disappearance of MSreactions. This character of MS was called habituation. Habituation effect would decreaseor even disappear with a long-term absence of stimulus. However, the periodical regularityand central mechanism of these MS characters still remains unclear.As we know, the vestibular system is the major receptor system of the outsideinformation of acceleration. Glutamate is a major excitatory neurotransmitter of primaryvestibular afferents and N-methyl-D-aspartate (NMDA) type ionotropic glutamatereceptors are abundantly localized in vestibular nucleus neurons. Many studies showed thatNMDA receptor-mediated LTP was the neural basis of learning and memory in the centralnervous system. Studies had also shown that NMDA receptor-mediated intracellular signaltransduction cascade mainly through the activation of NMDA receptors by glutamate, bycalcium/calmodulin-dependent kinase II (Ca2+/CaMKII) pathway, and ultimately activationof cAMP response element binding protein (CREB). Phosphorylation of CREB couldfurther induce the expression of immediate early gene c-fos and brain derived neurotrophicfactor (BDNF) gene and other new genes. In our previous study that Fos protein can beused as a specific molecular marker for activation of vestibular nucleus neurons and theglutamatergic vestibule-visceral projection pathway might be activated during FWLrotation. Moreover, a lot of studies had shown that many connections between thevestibular nucleus complexes were found, and activation of the vestibular system couldaffect hippocampal function and spatial memory. The aim of present study was designed toinvestigate the behavioral changes during repeated rotation treatment, and further examinewhether NMDA signaling pathway involves in motion sickness habituation andde-habituation in rat caudal vestibular nucleus and hippocampus, and finally examine theeffect of blocking the signaling pathway using pharmacological techniques on motion sicknesshabituation and de-habituation. 1.Animal model of motion sickness habituation and dehabituation1.1Animals and groupingAdult male Sprague–Dawley (SD) rats weighing220–250g were used in thisexperiment.1.1.1Long-time experimentTwenty male SD rats were used in this experiment and divided into rotation group andstatic control group (n=10). They were used to measure the number of fecal granule, thevolume of urine and the amount of kaolin and food intake during the1st,2nd,3rd, and4thhour of consecutive4-hour treatment. Another eighty male rats were randomly divided intoeight groups including rotation groups which received1h,2h,3h and4h rotationstimulation respectively and correspondent static control group without rotation (n=10).Spontaneous activity of each group was measured immediately after treatment.1.1.2Scopolamine experimentThirty male SD rats were used in this experiment. They were divided into threegroups: static control group, rotation control group, and free moving control group inwhich animals were maintained in individual cage without rotation and restraintstimulation (n=10).Sixty male SD rats were used in this experiment. They were randomly divided intosolvent control group, scopolamine I group (Sco0.5mg/kg) and scopolamine II group (Sco1.0mg/kg)(n=20), in which animals received solvent or scopolamine (i.g.) with the samevolume30minutes before each treatment session and then they were subdivided into staticand rotation groups, respectively (n=10in each subgroup).1.1.3Labyrinthectomy experimentForty male SD rats were used in this experiment. They were randomly divided intobilateral labyrinthectomy group and sham-lesioned group (n=20), in which animalsreceived operation4weeks before experiment started. Then they were subdivided intostatic and rotation groups, respectively (n=10in each subgroup).1.1.4Habituation experimentForty male rats were randomly divided into rotation treatment and static control group. (n=20). Animals in Rot group received daily2-hour static treatment for3days prior to thebeginning of the rotation sessions. Then they were rotated for2-hours per day at the sametime on the following days. Animals in static control group were treated similarly to theRot group except that they were not rotated. Defecation and urination responses and kaolinconsumption were measured daily during3-day prerotation and the following rotationsessions in all animals. To test the spontaneous activity during daily rotation sessions,another100male rats were used and assigned to5rotation groups receiving1,4,7,10and13daily2-hour rotation sessions, respectively and5corresponding Sta control groups(n=10).1.1.5De-habituation experimentIn this experiment, de-habituation treatment procedure was divided into threedifferent phases—2h/d repeated rotation to achieve habituation, no-rotation to getde-habituation, and re-rotation to test MS symptoms. At first, animals were rotated for2h/d and MS symptoms were measured daily (n=60). When the MS symptoms of animalsrecovered to normal level, rotation treatment ended temporally and animals were assignedto several subgroups which were re-rotated on day5,7,14,21,28and35after habituationachieved, respectively (n=10in each subgroup). In the meantime, another120animalsmatched in age and body weight with de-habituation experiment subgroups were dividedinto5rotation control and5static control groups which were rotated or not on thecorresponding re-rotation day (n=10in each group).1.2Labyrinthectomy experiment and Sham lesion experimentBilateral labyrinthectomy (BL) was performed under anesthesia4weeks beforeexperiment started. In brief, animals were initially anesthetized with sodium pentobarbital(40mg/kg, i.p.). Under an operating microscope, the mastoid was exposed by the retroauricular approach. Dental burr were then used to open the bony horizontal semicircularcanal. Then the membranous labyrinth was surgically removed with a small hook and thenthoroughly destroyed by injection of100%ethanol into the opened bony canal. Tympanicmembrane and ossicular chain remained intact during operation. At the end of surgery,antibiotic powder (Levofloxacin) was topically applied to the opened labyrinth to preventinfection and the temporal bone was sealed with dental cement. The operative wound wassutured and the animal was allowed to recover in the light. For the bilaterallabyrinthectomy, the second operation for controlateral labyrinth was performed24h after the first one. In sham-lesioned (SL) control animals, same procedures as above wereconducted excepted that only the bony horizontal semicircular canal was opened and innerear structure remained intact.1.3Rotation method and behavioral testEach conscious rat was rotated by a Forris-wheel like method. The rotation deviceused in the present experiment was modified based on that for cats by Crampton and Lucot.In brief, plexiglass containers suspended on a metal frame revolved about an axis parallelto the floor. Animals in static groups (Sta) were only enclosed in the restrainer for the sameperiod of time as rotation animals but not rotated. The consumption data of kaolin wascollected automated by the Comprehensive Lab Animal Monitoring System (CLAMS;Columbus Instruments, Columbus, OH) daily between10:00–11:00a.m. Spontaneousactivity test over the3min period was recorded using a computer-based event recorder andanalyzed by its software. This test was carried out immediately after the end of everyrotation or static treatment session.2. Effect of NMDA signaling pathway in vestibular nucleus neuronsduring habituation and de-habituation2.1Animals and groupingAdult male Sprague–Dawley (SD) rats weighing220–250g were used. Eighty animalswere used for western blot and real-time quantitative PCR (RT-PCR) test and randomlydivided into five rotation treatment (Rot) groups receiving1,4,7,10or13rotationsessions on a daily basis (2h/d), respectively, and five corresponding static control (Sta)groups (kept in the restrainer near the rotation device when Rot animals were being rotated)(n=8in each group). Forty eight animals were used for immunohistochemistry andimmunofluorescence study and divided into five Rot groups treated the same as above anda Sta group (kept in the restrainer for2h but not rotated)(n=8in each group).2.2Molecular biology experimentDuring habituation and de-habituation treatment procedure, the concentrations ofNMDA R1receptor (NR1), NMDA R2A/B receptor (NR2A/B), GABAAα1, calmodulinprotein kinaseIIα subunit (αCaMKII), phosphorylation of αCaMKII (p-αCaMKII), thenuclear transcription factor cAMP response element-binding (CREB), phosphorylation ofCREB (p-CREB) protein in the caudal vestibular nucleus were assessed by Western blot. Fos, Arc and brain-derived neurotrophic factor (BDNF) mRNA level in the caudalvestibular nucleus were assessed by real-time quantitative PCR. Fos expression in thecaudal vestibular nucleus were analyses by immunohistochemistry. During daily rotationtreatment sessions, co-expression of GABAAα1subunit with Arc in the caudal vestibularnucleus neurons was analyses by immunofluorescence.3. Effect of NMDA signaling pathway in hippocampal neurons duringhabituation and de-habituation3.1Animals and groupingAdult male Sprague–Dawley (SD) rats weighing220–250g were used. Eighty animalswere used for western blot and real-time quantitative PCR (RT-PCR) test and randomlydivided into five rotation treatment (Rot) groups receiving1,4,7,10or13rotationsessions on a daily basis (2h/d), respectively, and five corresponding static control (Sta)groups (kept in the restrainer near the rotation device when Rot animals were being rotated)(n=8in each group).Thirty animals were divided into five dehabituation groups receiving1,4,7,14or21no rotation sessions after habituation achieved, respectively, and a Sta group (n=5in eachgroup). One hundred and fifty animals were divided into five KN-93(KN) groupsreceiving2,4,6,8,10no rotation sessions after habituation achieved, and fivecorresponding sodium chloride control (SC) groups and five dehabituation control (dehab)groups (n=10in each group). On no rotation day, animals in KN and SC groups weremicroinjected KN-93and sodium chloride into the center of happocampus, respectively.3.2MicroinjectionRats were anesthetized with diethyl ether and placed in a rodent stereotaxic apparatus.The skin over the skull was incised and burr holes were drilled manually. Needles wereinserted through the burr holes, animals were bilaterally implanted with indwelling guidecannulae stereotaxically aimed center of the hippocampus (coordinates AP3.8mm；ML2.3mm；DV3.0mm from bregma point). Animals were allowed to recover from surgeryduring7days before submitting them to any other procedure. At the time of drug delivery,a infusion cannula was tightly ftted into the guides. Infusions (0.5μl/side) were carried outover60s, frst on one side and then on the other; the infusion cannula was left in place for60additional seconds to minimize backfow. 3.3Behaviour experimentRotation produce was same as produce1.4. De-habituation treatment procedure weredescribed in3.1. Fecal granule, total distance and total activity were detected after rotation.3.4Molecular biology experimentDuring habituation and de-habituation treatment procedure, the concentrations of NR1,NR2A/B, αCaMKII, p-αCaMKII, CREB, p-CREB protein in the hippocampus wereassessed by Western blot. Fos, Arc and BDNF mRNA level in the hippocampus wereassessed by real-time quantitative PCR.4. Statistical analysisStatistical analysis was performed using the SPSS v13.0statistical program.Two-factorial analysis of variance (ANOVA) was performed using General Linear Protocolto examine whether there were any significant effect of rotation, time and/or interactioneffect of rotation×time throughout the whole treatment process. Bonferroni post hoc testwas used to analyze the difference between Rot and Sta group, when a significant rotation×time interaction effect was obtained. The data for each Rot group were expressed as foldchange of the mean±SEM relative to the mean of the corresponding Sta group inhabituation, de-habituation, western blot and RT-PCR experiments.In behavioral response experiment, ANOVA and bonferroni post hoc test was used toanalyze the difference between Rot and Sta group. In immunohistochemistry experiment,one-way ANOVA combined with Dunnett-t test was performed to analyze the number ofFos-LI, GABAAα1-LI, GABAAα1/Arc-LI neurons and Arc/neuron in the caudalvestibular nuclei (MVe and SpVe). All data presented were expressed as mean±SEM.Statistical significance was judged at p<0.05.Results1.Animal model of motion sickness habituation and dehabituation1.1Effect of long time on MS behavior indexDuring4h rotation treatment, the number of fecal granule significantly increased inthe first and second hour (P<0.05) and decreased in the following third and fourth hourcompared with the static control group (P>0.05).24h kaolin consumption increasedthrough the4hours (P<0.05). Other indexes measured per hour showed no difference between rotation and static control data during the consecutive4h rotation (P>0.05). Inspontaneous activity test, the total activity and movement were significantly decreased in1h,2h rotation group compared with corresponding static control group (P<0.05), while itwas increased in3h and4h rotation group and showed no significant difference to staticgroup (P>0.05).1.2Effect of scopolamine on MS behavior indexAnti-MS medication pretreatment experiment was signed by using a single dose ofscopolamine at0.5or1.0mg/kg (i.g.). Both doses showed effect on decrease in defecationand24h kaolin consumption compared with solvent controls (P<0.05), and they evenrecovered to static control level. However, agent application (solvent or scopolamine)slightly increased the urine volume probably due to the dieresis effect of the solute(P<0.05). In spontaneous activity test, compared to the static control,2h rotation led tosignificant decrease in exploring behavior and total activity (P<0.05). In the meantime,scopolamine treatment at both doses attenuated the reduction effect on spontaneousactivity after rotation compared to the rotation control group.1.3Effect of labyrinthectomy on MS behavior indexIn sham operated animals, compared with static control group, the number of fecalgranules and24h kaolin consumption were increased, and the total activity and movementwas decreased significantly (P<0.05), and reached to the level of rotation control group(P>0.05). In bilateral labyrinthectomy (BL) group, no obvious change of MS symptomsand spontaneous activity was observed after rotation compared with static control group(P>0.05).1.4Habituation effect on MS behavior indexStatistical analysis found significant difference in number of fecal granules betweenRot and Sta groups from rotation session1to rotation session8(P<0.05), no differencewas found during9-14session (P>0.05). Statistical analysis revealed significant increase indaily24h kaolin consumption of Rot group from rotation session3to rotation session29(P<0.05), when compared with Sta group, while no difference was found when animalsreceived31rotation sessions (P>0.05). There is a significant decrease in total travelingdistance and total activity in animals receiving1and4rotation sessions when compared tocorresponding static controls (P<0.05). It recovered to static control level in animalsreceiving7,10and13rotation sessions (P>0.05). 1.5De-habituation effect on MS behavior indexAfter9days of2h/d rotation treatment, fecal granule recovered to base level inde-habituation (De-hab) group. It still remained at control level after5days withoutrotation, indicating that the animals maintained habituation state during these days(P>0.05). While, after7days without rotation, re-stimulation induced significant increasein fecal granule back to rotation control (Rot-con) level compared with static control group(P>0.05). Although, other behavior responses also showed some difference betweende-habituation and control groups on certain days after rotation paused, no character ortrend of de-habituation effect was observed. In spontaneous activity test, de-habituationeffect was observed in exploring behavior and total activity after21days without rotation,which recovered to rotation control level but lower than static control level indicated thatmotion sickness signs showed again.2. Effect of NMDA signaling pathway in vestibular nucleus neuronsduring habituation and de-habituation2.1Effect of daily rotation on NR1, NR2A/B and GABAAα1protein level andαCaMKII and CREB phosphorylation in the caudal vestibular nucleusIn rat caudal vestibular nucleus, statistical analysis found that GABAAα1andp-αCaMKII/αCaMKII increased significantly in animals receiving1or4rotation sessions(P<0.05) compared with corresponding Sta controls and reduced to baseline level after7sessions (P>0.05). Compared with the Sta control animals, p-CREB/CREB increased inanimals exposed to1,4,7or10rotation sessions (P<0.05) and reduced to baseline levelafter13sessions (P>0.05). There was no significant effect of rotation or time on NR1andNR2A/B level in Rot groups during the whole process during the13rotation sessions(P>0.05).2.2Effect of daily rotation on Fos, Arc and BDNF mRNA transcription in thecaudal vestibular nucleusIn rat caudal vestibular nucleus, statistical analysis revealed that compared with Stacontrol animals, c-fos mRNA increased in animals exposed to1and4rotation sessions(P<0.05). Arc mRNA decreased in animals exposed to1and4rotation sessions comparedwith corresponding Sta controls (P<0.05). No significant effect of rotation or time on BDNF mRNAlevel was found in Rot groups during the whole process (P>0.05).2.3Effect of daily rotation on Fos expression in the caudal vestibular nucleusOne-way ANOVA revealed that there was an overall difference in the amount ofFos-LI neurons in the MVe and the SpVe among Rot and Sta control group (P<0.01).Bonferroni post hoc analysis found that the amount of Fos-LI neurons increased in theMVe and the SpVe of animals receiving1,4or7rotation sessions (P<0.05) and reduced tobaseline in animals receiving10,13rotation sessions (P>0.05).2.4Co-expression of GABAAα1subunit with Arc in the caudal vestibularnucleus neurons during repeated rotation sessionOne-way ANOVA revealed that the amount of GABAAα1-LI and GABAAα1/Arc-LIneurons and Arc/neuron was significantly different between Rot and Sta control groups(P<0.01). Dunnett-t test analysis found that the number of GABAAα1-LI neurons in thecaudal vestibular nucleus were increased while GABAAα1/Arc-LI neurons and Arc/neuron were decreased in animals receiving1or4rotation sessions (P<0.05) andrecovered to Sta control level in animals receiving more than7rotation sessions (P>0.05).3. Effect of NMDA signaling pathway in hippocampal neurons duringhabituation and de-habituation3.1Effect of daily rotation on NR1and NR2A/B protein level and αCaMKIIand CREB phosphorylation in the hippocampusIn rat hippocampus, statistical analysis found that p-αCaMKII/αCaMKII increasedsignificantly in animals receiving13rotation sessions (P<0.05) compared withcorresponding Sta controls, and on day1,4,7,10, it had no change (P>0.05).P-CREB/CREB increased slowly in animals exposed to1,4,7or10rotation sessions(P>0.05) and increased significantly on day13(P<0.05) compared with the Sta controlanimals. There was no significant effect of rotation or time on NR1and NR2A/B level inRot groups during the whole process during the13rotation sessions (P>0.05).3.2Effect of daily rotation on Fos, Arc and BDNF mRNA transcription in thehippocampusIn rat hippocampus, there was no significant effect of rotation or time on Fos, Arc, BDNF mRNA level in Rot groups during the whole process during the13rotation sessions(P>0.05).3.3Effect of de-habituation on αCaMKII and CREB phosphorylation in thehippocampusIn rat hippocampus, statistical analysis found that p-αCaMKII/αCaMKII increasedsignificantly in animals after5,7,14day without rotation (P<0.05) compared withcorresponding Sta controls, and reduced to baseline level after21days (P>0.05).Compared with the Sta control animals, p-CREB/CREB increased in animals after1,4daywithout rotation (P<0.05) and reduced to baseline level after7days (P>0.05).3.4Effect of KN-93on behavior response of MS de-habituationAfter9days of2h/d rotation treatment, fecal granule recovered to base level inKN-93(KN) group, sodium chloride control (SC) group and de-habituation control (Dehab)group. As the result of1.5, fecal granule in SC and Dehab group still remained at controllevel after2,4,6days without rotation, indicating that the animals maintained habituationstate during these days (P>0.05). While, after8days without rotation, re-stimulationinduced significant increase in fecal granule back to rotation control level compared withstatic control group (P>0.05). Fecal granule in KN group increased after6days withoutrotation, indicating that de-habituation time was6days. In spontaneous activity test,de-habituation effect was observed in exploring behavior and total activity after6dayswithout rotation, which lower than dehab control group (P<0.05) indicated that motionsickness signs showed again.Conclusions1. In the present study, defecation during simulated motion sickness stimulation is asensitive MS specific symptom of rats. It is also suggested that counting the number offecal granule excreted during MS stimulation in rats can be an easy and reliable method forevaluating MS closely related characterizations including habituation and de-habituation.In the meantime, spontaneous activity test immediately after MS stimulation is also aneffective assay indicative of MS development. These indexes could be used to enhance theefficiency for judgment of motion sickness symptom. Pica, which was widely used as aspecie relevant paradigm of motion sickness in rodents by examining the kaolin intakeduring24hours after MS stimulation for many years, did not increase when the rats was firstly exposed to rotation, and not recover as rapidly as other behavioral response.Because of it, pica did not fit to research habituation and de-habituation of motionsickness.2. In the present study, after repeated exposure to MS stimulation, defecation andspontaneous activity response were gradually alleviated and almost returns to control levelon treatment day9and7, respectively. When the stimulation paused for about7and21days after habituation achieved, defecation and spontaneous activity response wasre-activated back to the pretreatment level indicative of the retrieval of the susceptibility toMS stimulation. Although these two indexes were not consistent with each otherthoroughly on the judgment of habituation and de-habituation progress, it might reflect theasynchronous response of the autonomic nerve system and cerebrum during MSstimulation. In conclusion, habituation time of rat was9days, and de-habituation time was21days.3. The present study showed that repeated rotation stimulation lead to change inNMDA receptor signaling and GABAAα1receptor expression in caudal vestibular nucleusneurons. During sickness phrase, αCaMKII and CREB activity and Fos mRNA levelincreased; GABAAreceptor α1subunit protein level increased and Arc mRNA leveldecreased. During habituation phrase, αCaMKII activity, Fos and Arc mRNA and GABAAα1protein level recovered back to control level. These results suggested that activation ofNMDA receptor and increment of GABAAα1receptor level in caudal vestibular nucleusassociate with motion sickness habituation development, and may provide a kind ofbiological basis for habituation of motion sickness.4. In the present study, αCaMKII and CREB activity increased during habituationphrase, and lasted to de-habituation phrase. Microinjection of CaMKII inhibitor KN-93could obviously advance de-habituation time of motion sickness. These results suggestedthat activation of NMDA receptor in rat hippocampus associated with the maintenance ofhabituation, and may play a part in habituation of motion sickness. |
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