快速眼动睡眠剥夺后大鼠海马CB-1受体mRNA表达及其与癫痫发病的相关性研究
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摘要
睡眠具有两种不同的时相状态,其一是脑电波呈现同步化慢波的时相,称为慢波睡眠(slow wave sleep,SWS),其二是脑电波呈现去同步化快波的时相,称为快速眼球运动(rapid eye movements,REM)睡眠。SWS与REM睡眠是两个互相转化的时相。REM睡眠对神经系统的成熟密切相关,对促进全身机能的恢复至关重要,有利于建立新的突触联系并促进学习记忆活动。睡眠剥夺(sleepdeprivation,SD)是睡眠障碍中常见症状,长时间睡眠剥夺使人体紧张、易疲劳,免疫机能紊乱,癫痫易发。近年国内外关于睡眠剥夺后脑组织生化及病理改变的研究较多,主要涉及氨基酸神经递质、一氧化氮、皮质醇、早期即刻基因表达等。大麻素CB-1受体是一种G-蛋白-耦联受体,它在海马区含量较高,可与内源性大麻素结合引导睡眠转换机制,能终止抽搐发作,对神经细胞具有保护作用,在癫痫发病机制中可能起到自身稳定调节的作用。但国内外对大麻素CB-1受体、睡眠剥夺及癫痫三者之间的关系研究未见报道。
内源性大麻素通过与CB-1受体结合可抑制突触活性,提高癫痫阈值而起到抗癫痫作用。本课题通过建立睡眠剥夺模型,观察大鼠的行为和神经元超微结构改变,大麻素CB-1受体mRNA表达的动态变化,及其表达高低与癫痫发病
BackgroundSleep has two different stages,one is slow wave sleep (SWS) the other is rapid eye movements sleep(REM). REM sleep is good for the development of the neurons. Sleep deprivation(SD) can cause nervouse,tiresome, immune- disorder and epilepsy.The research of sleep deprivation ; cannabinoid receptor 1 and epilepsy has not been discussed nowday. In this study we observed the expression of cannabinoid receptor 1 after rapid eye movement sleep deprivation in rat hippocampus and its contribution to epilepsy. Our goal was to determine the effect of sleep deprivation on neurons and how CB-1 receptor play an important role in epilepsy.Objects and MethodsTotally 65 Sprague-Dawley male rats weighing 220-250g were involved in the experiment and divided randomly into two groups,as epilepsic group and unepilepsic
group.The rats of unepilepsic group were divided randomly as cage control(CC),tank control(TC)and the sleep deprivation group(SD).The SD and TC group animals were sacrificed at the end of ld,3d,5d sleep deprivation periods respectively.The rats of epilepsic group were also divided randomly as tank control(TC)and the sleep deprivation group(SD).The modified multiple plateform methods(MMPM)were established for the rapid eye movement(REM)sleep deprivation. CB-1 receptor mRNA were measured by using reverse transcription-polymerase chain reaction(RT-PCR). The different stage of ultrostructure were observed through electro-microscope. Using pentetrazole(PTZ) to induce convulsion and observe the expression of CB-1 receptor after seizure.The data was presented as means and standard deviations (x ± s) .Independent samples T-test was performed among groups.Multiple linear regression analysis was performed in epilepsic groups.ResultsAfter 1 day sleep deprivation the rats became irritating.Three days later the phenomenon was more serious.At the end of 5 days the rats calmed down. Neurons apoptosis were found in the SD3d and SD5d groups through electro-microscope.The SD1d group displayed increased expression of CB-1 receptor as compared with the TC1d group(P<0.05) while the SD3d group displayed minimum expression. The SD5d group did not experience significant changes as compared with the TC5d group(P > 0.05). After injecting PTZ we found the SD3d group suffered a serious convulsion.furthermore 2 of which developed to epileptic state and the expression of CB-1 receptor was the lowest among 3 groups(P<0.05).However the TC group did not experience a significant increased or decreased expression of CB1 receptor.
DiscussionOur findings indicated that sleep deprivation is a bad stress to the brain which cause neurons apoptosis and functional incapacitation. Suffered from sleep deprivation the expression of cannabinoid receptor 1 mRNA is increased in early stage.With the sleep deprivation prolonging CB1 receptor becomes decomposed and the expression decreased to the lowest level.Continusly, owing to inverse feedback it keeps transient balance for a short time.The data suggested that cannabinoid receptor 1 plays an important protective role in epilepsy. CB1 receptor activation is known to induce hyperpolarization of neuronal membranes,mainly by increasing K+ and decreasing Ca2+ conductance.Such a hyperpolarization,caused by an autocrine or paracrine activation of CB1 receptors by endocannabinoids would decrease the L-glutamate release evoked during excitotoxicity. CB1 receptors mediate protection against excitotoxicity not only by dampening the neuronal excitability of pyramidal neurons but also by inducing intracellular cascades,including ERK phosphorylation and the expression of immediate early genes(IEGs) that code for transcription factors(c-fos and zif 268) and neurotrophins(such as BDNF).Based on our findings we guess the modulation of the expression of CB1 receptor or its agonists may have an anticonvulsant efficiency which may illuminate novel therapeutic targets for the treatment of epilepsy.
Conclusions1. Sleep deprivation can cause brain dysfunction and modify the expression of CB1 receptor mRNA.2. The down-regulation of th
内源性大麻素通过与CB-1受体结合可抑制突触活性,提高癫痫阈值而起到抗癫痫作用。本课题通过建立睡眠剥夺模型,观察大鼠的行为和神经元超微结构改变,大麻素CB-1受体mRNA表达的动态变化,及其表达高低与癫痫发病
BackgroundSleep has two different stages,one is slow wave sleep (SWS) the other is rapid eye movements sleep(REM). REM sleep is good for the development of the neurons. Sleep deprivation(SD) can cause nervouse,tiresome, immune- disorder and epilepsy.The research of sleep deprivation ; cannabinoid receptor 1 and epilepsy has not been discussed nowday. In this study we observed the expression of cannabinoid receptor 1 after rapid eye movement sleep deprivation in rat hippocampus and its contribution to epilepsy. Our goal was to determine the effect of sleep deprivation on neurons and how CB-1 receptor play an important role in epilepsy.Objects and MethodsTotally 65 Sprague-Dawley male rats weighing 220-250g were involved in the experiment and divided randomly into two groups,as epilepsic group and unepilepsic
group.The rats of unepilepsic group were divided randomly as cage control(CC),tank control(TC)and the sleep deprivation group(SD).The SD and TC group animals were sacrificed at the end of ld,3d,5d sleep deprivation periods respectively.The rats of epilepsic group were also divided randomly as tank control(TC)and the sleep deprivation group(SD).The modified multiple plateform methods(MMPM)were established for the rapid eye movement(REM)sleep deprivation. CB-1 receptor mRNA were measured by using reverse transcription-polymerase chain reaction(RT-PCR). The different stage of ultrostructure were observed through electro-microscope. Using pentetrazole(PTZ) to induce convulsion and observe the expression of CB-1 receptor after seizure.The data was presented as means and standard deviations (x ± s) .Independent samples T-test was performed among groups.Multiple linear regression analysis was performed in epilepsic groups.ResultsAfter 1 day sleep deprivation the rats became irritating.Three days later the phenomenon was more serious.At the end of 5 days the rats calmed down. Neurons apoptosis were found in the SD3d and SD5d groups through electro-microscope.The SD1d group displayed increased expression of CB-1 receptor as compared with the TC1d group(P<0.05) while the SD3d group displayed minimum expression. The SD5d group did not experience significant changes as compared with the TC5d group(P > 0.05). After injecting PTZ we found the SD3d group suffered a serious convulsion.furthermore 2 of which developed to epileptic state and the expression of CB-1 receptor was the lowest among 3 groups(P<0.05).However the TC group did not experience a significant increased or decreased expression of CB1 receptor.
DiscussionOur findings indicated that sleep deprivation is a bad stress to the brain which cause neurons apoptosis and functional incapacitation. Suffered from sleep deprivation the expression of cannabinoid receptor 1 mRNA is increased in early stage.With the sleep deprivation prolonging CB1 receptor becomes decomposed and the expression decreased to the lowest level.Continusly, owing to inverse feedback it keeps transient balance for a short time.The data suggested that cannabinoid receptor 1 plays an important protective role in epilepsy. CB1 receptor activation is known to induce hyperpolarization of neuronal membranes,mainly by increasing K+ and decreasing Ca2+ conductance.Such a hyperpolarization,caused by an autocrine or paracrine activation of CB1 receptors by endocannabinoids would decrease the L-glutamate release evoked during excitotoxicity. CB1 receptors mediate protection against excitotoxicity not only by dampening the neuronal excitability of pyramidal neurons but also by inducing intracellular cascades,including ERK phosphorylation and the expression of immediate early genes(IEGs) that code for transcription factors(c-fos and zif 268) and neurotrophins(such as BDNF).Based on our findings we guess the modulation of the expression of CB1 receptor or its agonists may have an anticonvulsant efficiency which may illuminate novel therapeutic targets for the treatment of epilepsy.
Conclusions1. Sleep deprivation can cause brain dysfunction and modify the expression of CB1 receptor mRNA.2. The down-regulation of th
引文
[1] Suchecki D,Tufik S.Social stability attenuates the stress in the modified multiple platform method for paradoxical sleep deprivation in the rat.Physiol Behav,2000,68:309-316.
[2] Racine R.J., Modification of seizure activity by electrical-stimulation. Ⅱ. Moter seizure, EEG Clin. Neurophysiol. 1972, 32:281-294.
[3] Laureys S,Peigneux P, Perrin F, et al.Sleep and motor skill learning.Neuron,2002, 35:5-7.
[4] Gonzalez-Ortiz M,Martinez-Abundis E,Balcazar-Munoz BR,et al.Effect of sleep deprivation on insulin sensitivity and cortisol concentration in healthy subject.Diabetes Nutr Metab,2000,13:80-83.
[5] 黄建欧,田国红,赵忠新,等.快速眼动睡眠剥夺后大鼠皮质及海马HSP70表达的研究.中华神经科杂志,2004,37(4):331-334.
[6] 吴兴曲,杨来启,李栓德,等.睡眠剥夺后大鼠海马生化及病理变化.中国心理卫生杂志,2003,17(2):77-79.
[7] Majumdar S, Mallick B.N.Incraese levels of tyrosine hydroxylase and glutamic acid decarboxylase in locus coeruleus neurous after rapid eye movement sleep deprivation in rats. Neuroscience Letters,2003,338:193-196.
[8] Satvinder Kaur, Manoj Panchal, Mohd.Faisal,et al.Long term blocking of GABA-A Receptor in locus coeruleus by bilateral microinfusion of picrotoxin reduced rapid eye movement sleep and increased brain Na-K ATPase activity in freely moving normally behaving rats.Behavioural Brain Research,2004,151: 185-190.
[9] Lopez-Rodriguez F, Milson CL, Maidment NT, et al.Total sleep deprivation increases extracellular serotonin in the rat hippocampus. Neuroscience, 2003, 121:523-530.
[10] Terao A, Greco M.A, Davis R.W,et al.Region-specific changes in immediate early gene expression in response to sleep deprivation and recovery sleep in the mouse brain. Neuroscience,2003,120:1115-1124.
[11] Giovanni Marsicano,Sharon Goodenough,Krisztina Monory,et al.CBl cannabinoid receptors and on-demand defense against excitotoxicity. Science, 2003, 302:84-88.
[12] Melisa J.Wallace,Robert E.Blair,Katherine W.Falenski,et al.The endogenous cannabinoid system regulates seizure freguency and duration in a model of temporal lobe epilepsy.J Pharmacol Exp Ther,2003,307:129-137.
[13] Navarro.L, martinez-vargas.M, Murillo-rodriguez.E,et al.Potential role of the cannabinoid receptor CB1 in rapid eye movemente sleep rebound. Neuroscience, 2003, 120: 855- 859.
[14] Martinez- vargas M, Murillo-rodriguez E, Gonzalez-rivera R,etal.Sleep modulates cannabinoid receptor 1 expression in the pons of rats. Neuroscience, 2003,117: 197-201.
[15] Benjamin J.Whalley,Jonathan D.Wilkinson,Elizabeth M.Williamson,et al.A novel component of cannabis extract potentiates excitatory synaptic transmission in rat olfactory cortex in vitro.Neuroscience Letters,2004,365:58-63.
[16] Shaffery J.P, Sinton C.M, Bissette G,et al. Rapid eye movemente sleep deprivation modifies expression of long-term potentiation in visual cortex of immature rats. Neuroscience,2002,110:431 -443.
[17] Ramanathan L, Gulyani S,et al,Pathology in sleep deprivation decrease superoxide dismutase activity in rat hippocampus and brainstem. Neuroreport, 2002,13:1387-1390.
[2] Racine R.J., Modification of seizure activity by electrical-stimulation. Ⅱ. Moter seizure, EEG Clin. Neurophysiol. 1972, 32:281-294.
[3] Laureys S,Peigneux P, Perrin F, et al.Sleep and motor skill learning.Neuron,2002, 35:5-7.
[4] Gonzalez-Ortiz M,Martinez-Abundis E,Balcazar-Munoz BR,et al.Effect of sleep deprivation on insulin sensitivity and cortisol concentration in healthy subject.Diabetes Nutr Metab,2000,13:80-83.
[5] 黄建欧,田国红,赵忠新,等.快速眼动睡眠剥夺后大鼠皮质及海马HSP70表达的研究.中华神经科杂志,2004,37(4):331-334.
[6] 吴兴曲,杨来启,李栓德,等.睡眠剥夺后大鼠海马生化及病理变化.中国心理卫生杂志,2003,17(2):77-79.
[7] Majumdar S, Mallick B.N.Incraese levels of tyrosine hydroxylase and glutamic acid decarboxylase in locus coeruleus neurous after rapid eye movement sleep deprivation in rats. Neuroscience Letters,2003,338:193-196.
[8] Satvinder Kaur, Manoj Panchal, Mohd.Faisal,et al.Long term blocking of GABA-A Receptor in locus coeruleus by bilateral microinfusion of picrotoxin reduced rapid eye movement sleep and increased brain Na-K ATPase activity in freely moving normally behaving rats.Behavioural Brain Research,2004,151: 185-190.
[9] Lopez-Rodriguez F, Milson CL, Maidment NT, et al.Total sleep deprivation increases extracellular serotonin in the rat hippocampus. Neuroscience, 2003, 121:523-530.
[10] Terao A, Greco M.A, Davis R.W,et al.Region-specific changes in immediate early gene expression in response to sleep deprivation and recovery sleep in the mouse brain. Neuroscience,2003,120:1115-1124.
[11] Giovanni Marsicano,Sharon Goodenough,Krisztina Monory,et al.CBl cannabinoid receptors and on-demand defense against excitotoxicity. Science, 2003, 302:84-88.
[12] Melisa J.Wallace,Robert E.Blair,Katherine W.Falenski,et al.The endogenous cannabinoid system regulates seizure freguency and duration in a model of temporal lobe epilepsy.J Pharmacol Exp Ther,2003,307:129-137.
[13] Navarro.L, martinez-vargas.M, Murillo-rodriguez.E,et al.Potential role of the cannabinoid receptor CB1 in rapid eye movemente sleep rebound. Neuroscience, 2003, 120: 855- 859.
[14] Martinez- vargas M, Murillo-rodriguez E, Gonzalez-rivera R,etal.Sleep modulates cannabinoid receptor 1 expression in the pons of rats. Neuroscience, 2003,117: 197-201.
[15] Benjamin J.Whalley,Jonathan D.Wilkinson,Elizabeth M.Williamson,et al.A novel component of cannabis extract potentiates excitatory synaptic transmission in rat olfactory cortex in vitro.Neuroscience Letters,2004,365:58-63.
[16] Shaffery J.P, Sinton C.M, Bissette G,et al. Rapid eye movemente sleep deprivation modifies expression of long-term potentiation in visual cortex of immature rats. Neuroscience,2002,110:431 -443.
[17] Ramanathan L, Gulyani S,et al,Pathology in sleep deprivation decrease superoxide dismutase activity in rat hippocampus and brainstem. Neuroreport, 2002,13:1387-1390.