出生前后慢性铝暴露对年轻大鼠海LTP及细胞内Ca~（2+）浓度和CaMKⅡ表达的影响
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摘要
前言
铝是地壳中含量最丰富的元素之一,大量蓄积可产生神经毒性作用。临床观察长期肾透析的患者脑内铝的蓄积是正常人的15倍,患者最终出现视觉、记忆、注意力和额叶功能的多发性、联合性障碍。国内外流行病学调查和研究表明,铝易致神经元损伤,引起智力和认知能力下降等学习和记忆方面的缺欠。目前动物实验已证实,铝暴露可致大鼠痴呆,其表现为学习和记忆的行为障碍。
海马是学习和记忆的关键脑区,海马长时程增强(long-term potentiation,LTP)是NMDA受体依赖性突触传递效能的持续性增强,是脑学习和记忆功能在突触水平的研究模型和细胞基础。因此研究铝暴露对LTP及与其突触机制有关的各项生化指标的影响,有助于从突触和蛋白分子水平阐明铝损害脑学习和记忆功能的作用机制。目前虽然铝对LTP损害作用的观察很多,但铝对LTP损害作用的突触机制尚未完全阐明。
母体期和断乳后发育期是脑发育的重要阶段。在此阶段如果遭受了铝暴露,是否会影响其发育,特别是智力和认知能力的发育,这是一个关系到人类健康的重要问题,有关此阶段铝对学习和记忆及LTP影响的报道较少,因此有必要对其进行深入研究。本文拟从母体期和断乳后发育期慢性铝暴露对年轻大鼠的学习和记忆行为、海马LTP和细胞内钙([Ca~(2+)]_i)、钙调蛋白依赖性激酶Ⅱ(CaMKⅡ)的影响的角度,对此问题作以初步探讨,为揭示铝暴露损害脑学习、记忆功能的神经机制提供资料。
材料与方法
1、动物分组与染毒
健康Wistar大鼠45只,体重250 g左右,雌雄2∶1。适应饲养环境1 w后,随机分为3组:对照组、低剂量组和高剂量组,每组15只(雌雄2∶1)。对照组:饮用蒸馏水;低剂量组:饮用含0.2%AlCl_3的蒸馏水溶液(用0.2%-Al表示);高剂量组:饮用含0.4%AlCl_3的蒸馏水溶液(用0.4%-Al表示)。各组自染毒始即分别合笼,子代鼠出生后通过母乳继续染毒,断乳后(生后21天)各组子代鼠对应分别以蒸馏水或母鼠染毒剂量的AlCl_3溶液喂养,至子鼠生后90天。然后,按原母鼠染毒的剂量分组,每窝取1～2只子鼠(雌雄各半),组成子代各实验组,以蒸馏水喂养。动物室温度18℃～23℃,相对湿度45～55%。
2、脑组织及血液中铝含量测定
准确称取0.1～0.5 g的脑组织或准确吸取0.2～0.5 ml的全血于石英烧杯中,加5～8 ml混酸,同时做空白对照。采用原子吸收石墨炉法测定脑铝和血铝含量。
3、学习和记忆的行为学测定
测试方法为跳台法(即步下试验,Step-down)。学习行为训练:将大鼠放入跳台仪内适应3 min后训练其找到平台三次,大鼠跳下平台时,立即通电给予持续电刺激(36 V)并开始计时,记录5 min内大鼠受到电刺激第一次跳上平台躲避电击的反应时间(Escape latency,EL)以及受到电击的次数(Number of errors)。24 h后进行记忆保持测试:记录大鼠置于平台到第1次跳下平台的时间(Step-downlatency,SDL)和5 min内受到电击的次数(错误次数)。
4、电生理LTP测定
乌拉坦麻醉后,将动物头部固定于脑立体定位仪上,在Schaffer侧枝部位(坐标:前囟后3.3 mm;旁开3.8 mm;皮层下3.8 mm)插入刺激电极。将记录玻璃微电极插入海马CA1区部位(坐标:前囟后3.3 mm;旁开1.5 mm;电极尖端先抵皮层表面,然后逐步推进)行细胞外记录。找到稳定的群体锋电位(populationspike,PS)后,首先记录30 min,每分钟给予一次单脉冲刺激所诱发的PS。然后观察给予同样强度及波宽的的短串高频条件刺激后,每分钟给予一次单脉冲检验刺激所诱发的PS的幅值变化。
5、海马细胞内游离钙的测定
心脏采血并取脑,分离出海马组织并去除脑膜血管,制备成单细胞悬液,然后Fura-2/AM避光孵育负载,调激发波长340 nm,发射波长510 nm,采用日立F-3000型荧光双波长分光光度计测定。
6、海马钙调蛋白依赖性激酶Ⅱ(CaMKⅡ)的表达
实验后大鼠立即断头取脑分离并取双侧海马组织后按体积比为1:6-1:9放到预冷的裂解缓冲液中。4℃超声粉碎后离心30min,取上清分装。按照常规Westernblot方法分离蛋白质。将蛋白印迹显影图扫描,再利用ChemiImager 5500 V 2103图像分析软件对实验结果(测定目标带单位密度)进行分析。
7、统计学处理
实验所得数据资料用(?)+s表示,组间资料统计用单因素方差分析(ANOVA)检验,行为学数据不服从正态分布,采用秩和检验。
实验结果
1、各组大鼠血铝和脑铝含量的比较
随着铝暴露剂量的升高,铝暴露组大鼠的血铝、脑铝含量逐渐升高,且均显著高于对照组(P＜0.05 or P＜0.01),且两铝暴露组间差异也显著(P＜0.05)。
2、大鼠的学习记忆能力比较
与对照组相比,学习行为表现为第一次跳上平台的时间(反应时间)均明显长(P＜0.01),5 min内受到电击次数显著增多(P＜0.01)。记忆行为则表现为第一次跳下平台的潜伏期明显缩短(P＜0.01),5 min内错误次数明显增加(P＜0.01),且两铝暴露组间亦有显著性差异(P＜0.01)。
3、PS幅值的变化情况
随着铝暴露剂量的升高,高频刺激后PS幅值增强率依次降低,两铝暴露组与对照组相比,显著降低(P＜0.01),但两暴露组之间差异不显著(P＞0.05)。
4、海马神经元的细胞内[Ca~(2+)]i比较
随着铝暴露剂量的增加,海马神经元的细胞内Ca~(2+)浓度([Ca~(2+)]_i)逐渐降低。两铝暴露组与对照组相比均降低,0.2%-Al组与对照组的差异具有显著意义(P＜0.05),0.4%-Al组与对照组的差异则具有显著意义(P＜0.01),且0.2%-Al组与0.4%-Al组之间的差异也具有显著意义(＜0.05)。
5、海马CaMKⅡ的表达
与对照组相比,两铝暴露组海马CaMKⅡ的表达明显降低,具有统计学意义(P＜0.01),并且0.2%-Al组和0.4%-Al组之间亦有显著性差异(P＜0.01)。
讨论
出生前后(孕期、哺乳期、断乳后发育期)慢性铝暴露的年轻大鼠,与对照组相比其血铝和脑铝含量明显增高,LTP的增强率明显下降,学习行为表现为第一次跳上平台的时间(反应时间)均明显长,5 min内受到电击次数显著增多。记忆行为则表现为第一次跳下平台的潜伏期明显缩短,5 min内错误次数明显增加,表明母体的铝暴露可通过胎盘和血脑屏障进入子代鼠体内,造成铝在子代体内蓄积,特别是脑内的蓄积,进而损害了子代鼠的学习和记忆能力。本实验同时观察到,各组海马神经元[Ca~(2+)]_i和CaMKⅡ的表达也降低,提示出生前后连续慢性铝暴露年轻大鼠学习记忆能力下降的可能原因之一是铝干扰了海马神经元细胞内Ca~(2+)稳态平衡并进一步抑制了CaMKⅡ的表达。
有研究表明,铝可阻断电压依赖性钙通道(voltage-dependent calcium channels,VDCCs);和抑制磷脂酶C(phospholipase C,PLC)催化的磷脂酰二磷酸肌醇(phosphatidylinositol-4,5-diphosphate,PIP_2)的水解,造成作为第二信使的三磷酸肌醇(IP_3)和二酰甘油(DG)生成减少,进而影响了Ca~(2+)的内流和内质网钙库中Ca~(2+)的释放。
铝一方面降低了细胞内Ca~(2+)浓度,另一方面铝与钙竞争,抑制了钙与CaM(钙调蛋白)结合性,并使其构型改变,阻断了Ca~(2+)依赖性CaMKⅡ的作用,干扰了磷酸化过程。同时铝对CaMKⅡ参与LTP诱导与维持过程中所需的NMDA受体和AMPA受体等也有影响,因而进一步抑制了CaMKⅡ的表达。
综上所述,有理由认为,铝影响学习记忆能力的可能原因之一是其可通过多种途径引起细胞内Ca~(2+)浓度降低,并进一步抑制了CaMKⅡ的表达。
结论
(1)母体期和断乳后发育期慢性铝暴露的大鼠,与对照组相比,PS幅值增强率降低,提示该暴露损害了大鼠LTP的诱导与维持;
(2)母体期和断乳后发育期慢性铝暴露的年轻大鼠与对照组相比,学习行为表现为第一次跳上平台的时间(反应时间)均明显延长,5 min内受到电击次数显著增多。记忆行为则表现为第一次跳下平台的潜伏期明显缩短,5 min内错误次数明显增加,提示铝暴露损害了大鼠的学习和记忆行为;
(3)铝损害LTP和对神经元细胞的损伤的可能机制之一是铝造成了海马中的细胞内Ca~(2+)浓度降低;
(4)母体期和断乳后发育期慢性铝暴露可使大鼠神经元细胞内CaMKⅡ的表达受到抑制。
Introduction
Aluminum is one of the most abundant element on earth.It has neurotoxicity if much of that accumulate in human's body.Evidence of epidemiology has shown that Al can impair neuron and induce learning and memory deficiency.In experimentally induced dementia animal models,it has been demonstrated that there are a reduced capacity of learning and memory and the pathological change which is similar with Alzheimer's disease after Al exposure.
The hippocampus is the most important encephalic region relative to function of learning and memory.Hippocampal long-term potentiation(LTP)is NMDA receptor-dependent persistent enhancement of efficacy in synaptic transmission,it is reputed that LTP represents the most intensively studied synaptic model and cellular basis of learning and memory in the mammalian brain.So investigating the effect of aluminum exposure on LTP and the biochemical indicators relative to the LTP synaptic mechanism will help to elucidate the mechanism of aluminum damaging the learning and memory on synaptic and proteinic level.
Although Al has been reported to impair LTP following administration in vivo and in vitro,the underlying mechanisms of Al action on LTP are still unknown.The stage of maternal and postweaning is a very important period during brain development. When the brain is exoposed to Al during this period,whether its development especially the intelligence and learning abilities will be affected,which is an important matters concerned with human's health.There is little reports about the effect of Al on learning and memory and LTP of this stage,so it is necessary for us to have a deep research.
The present study has observed the effect of aluminum exposure during maternal and postweaning period on the offspring's learning and memory and the Ca~(2+), CaMKⅡof hippocampus,hope to elucidate the synaptic mechanisms of the impairs of Al exposure on the learning and memory.
Materials and methods
1.Group and exposure
Healthy adult Wistar rats(280-320)g were exposed to aluminum through drinking 0%(distilled water)or the concentration of Al~(3+)were 0.2 g/100ml(2 g·L~(-1))(represented by 0.2%-Al)and 0.4g/100ml(4g·L~(-1))(represented by 0.2%-Al)aluminum chloride(AlCl_3) solution,respectively,from duration of pregnancy to postnatal 90 days.Their offspring were distributed into three experimental groups:a controls group;two exposed groups (0.2%-Al group and 0.4%-Al group).And the room temperature is 18℃～23℃,relative humidity is 45%～55%.
2.Brain and blood Al determinations
The brain and blood aluminum concentrations were determined by atomic absorption plumbago.Weigh 0.1～0.5g brain tissue or take suction 0.2～0.5ml whole blood,add 5～8ml violet acid,blank together.
3.Learning and memory behavioral testing
The step-down test was used to monitor the learning and memory ability.Learning ability test:put the rat into the jumping fiat instrutment to accommodate for 3 minuts, then train them to find the flat three times,when rat jump off the flat,give them a electric stroke(36 V)and begin to record the time,recording the escape latency and the number of errors in 5 min.24 hours later,memory ability test:recording the step-down latency and the number of errors in 5min.
4.Electrophysiological recordings
The extracellular micropipette recording technique was used to recording electrophysiological change.Wistar rats were anaesthetized with 20%urethane(6.5 ml/kg,i.p.)and their heads were fixed in a stereotaxis instrument.Population spike(PS) was evoked by electrical stimulation of Schaffer Collateral pathway form the CA3 to the CA1 region of hippocampus using a concentric bipolar stimulating electrode located at the area CA3 of Schaffer collateral(3.8mm posterior to bregma,3.8mm lateral to the midline,3.8mm under cortex).Glass micropipettes filled with 3 M KCl were placed at the CA1 region of hippocampus(3.3mm posterior to bregma;1.5mm lateral to the midline;on the surface of cortex)and lowered into the area CA1 for recording filed potentials.After the response had stabililized in each rat,30 min baseline was recorded.Stimuli were given every minute(all responses were averaged for each date point).A high frequency stimulation(HFS)(100 Hz,5s)was delivered at same intensity.Testing with single pulses was continued after the HFS was delieved.
5.Intracellular Ca2+ concentration in hippocampus
Intracellular Ca~(2+)concentration in hippocampal cells was measured by the technique of Fura-2/AM calcium ions fluorescence indicator in SPF.The excitation wave is 340nm and the emission wave is 510nm.
6.The expression of CaMKⅡin hippocampus
Dislodged the hippocampus into cold lysate by 1:6-1:9 of volume ratio.Pulverized by transonic wave at 4℃and centrifuged in 12000 g 1 hour,then separated the supernatant.According to the technique of ChemiImager 5500 V 2103 picture analysis software,measured the expression of CaMKⅡin hippocampus.
7.statistical analysis
Experimental values were indicated as the mean±SD.Interclass data were tested by ANOVA.The results of behavioral testing are not normal distribution so by rank sum test.
Results
1.The comparison of blood and brain aluminum
The aluminum concentration in blood and brain tissue was significantly higher than the control group(P＜0.05 or P＜0.01)and increased with exposure dose. Furthermore,the difference between the two exposed group was statistically significant.
2.The comparison of learning and memory behavioural test
The behavioural data showed:the escape latency was remarkably prolonged,the step-down latency was remarkably decreased,and the number of errors both in learning and memory was significantly increased in the Al~(3+)exposed groups(P＜0.01 or P＜0.05) as compared to the control group.Furthermore,the difference between the two exposed group was statistically significant.
3.The comparison of electrophysiological recording results
With the increase of exposure dose,the PS magnitude reduced evidently aider HFS. As compared to the control group,the two exposed groups were significant reduced, but the difference between the two exposed groups was statistically non-significant.
4.The comparison of intracellular calcium of hippocampal cells
The hippocampus intracellular Ca~(2+)concentration was reduced in 0.2%-Al group as well as 0.4%-Al group,this reduction in 0.2%-Al group was statistically significant (P＜0.05),but was significantly decreased in 0.4%-Al exposed group(P＜0.01).The difference between 0.2%-Al and 0.4%-Al group was significantly(P＜0.05).
5.The expression of CaMKⅡin hippocampus
Compared to the control group,the expression of CaMKⅡin hippocampus were significantly reduced in 0.2%-Al group as well as 0.4%-Al group(P＜0.01).
Discussion
The young rats of Al exposure from prenatal to postnatal(maternal,pregnancy and postweaning)period could increase the level of Al in blood and brain,evidently decrease the amplitude of PS afer HFS,prolong remarkably the escape latency, shortened the latent period of jumping off the flat,increase the erring times during the 5 minutes in learning and memory behavioural test,it express that the maternal and postweaning period of Al exposure impaired cognitive function of the offspring through placenta and blood brain barrierand resulted in the accumulate of Al in thebrain of the offspring,impaired the offspring's learningand memory ability.In this study,the result also showed the decrease of intracellular Ca~(2+)and the expression of CaMKⅡin hippocampus,this suggested one of the reasons that maternal and postweaning chronic aluminum impaired the offspring's learning and memory ability is disturbing the intracellular Ca~(2+),balance,and further inhabit the expression of the CaMKⅡ.
The present results also showed that maternal and postweaning chronic Al exposure could decrease the intracellular Ca~(2+)concentration in hippocampus,which was dose-dependent.Some reports showed that Al meight interfere with[Ca~(2+)]_i by block the VDCCs;inhibited the expression of NMDA receptor in hippocampus;IP_3 decreaseaffected the Ca~(2+)inflowing and degraded the intracellular Ca~(2+)release from calcium reservoir pool.
In one aspect that Al decreased intracellular Ca~(2+)concentration,another Al competed with Ca~(2+)to integrate with CaM,moreover,altered it's configuration then interrupted the contribution of Ca~(2+)/CaMKⅡ,interfered with phosphorylation cours.At same time Al also affected NMDA receptor and AMPA receptor,consequently inhibited the expression of CaMKⅡ.
In a word,Al affected Ca~(2+)concentration and the expression of CaMKⅡin hippocampus by many means which played an important role in LTP.It's needed to be further investigations.
Conclusion
1.Chronic Al exposure from prenatal to postnatal period can impair the behaviour of the learning and memory in young rats;
2.Chronic Al exposure from prenatal to postnatal can make the amplitude of PS decrease,impaired the induction and maintenance of LTP in young rats.
3.One of the probable mechanisms of impairing LTP may be the decrease of [Ca~(2+)]i in hippocampus.
4.Another probable mechanisms of impairing LTP may be the decrease of CaMKⅡexpression by Al.
铝是地壳中含量最丰富的元素之一,大量蓄积可产生神经毒性作用。临床观察长期肾透析的患者脑内铝的蓄积是正常人的15倍,患者最终出现视觉、记忆、注意力和额叶功能的多发性、联合性障碍。国内外流行病学调查和研究表明,铝易致神经元损伤,引起智力和认知能力下降等学习和记忆方面的缺欠。目前动物实验已证实,铝暴露可致大鼠痴呆,其表现为学习和记忆的行为障碍。
海马是学习和记忆的关键脑区,海马长时程增强(long-term potentiation,LTP)是NMDA受体依赖性突触传递效能的持续性增强,是脑学习和记忆功能在突触水平的研究模型和细胞基础。因此研究铝暴露对LTP及与其突触机制有关的各项生化指标的影响,有助于从突触和蛋白分子水平阐明铝损害脑学习和记忆功能的作用机制。目前虽然铝对LTP损害作用的观察很多,但铝对LTP损害作用的突触机制尚未完全阐明。
母体期和断乳后发育期是脑发育的重要阶段。在此阶段如果遭受了铝暴露,是否会影响其发育,特别是智力和认知能力的发育,这是一个关系到人类健康的重要问题,有关此阶段铝对学习和记忆及LTP影响的报道较少,因此有必要对其进行深入研究。本文拟从母体期和断乳后发育期慢性铝暴露对年轻大鼠的学习和记忆行为、海马LTP和细胞内钙([Ca~(2+)]_i)、钙调蛋白依赖性激酶Ⅱ(CaMKⅡ)的影响的角度,对此问题作以初步探讨,为揭示铝暴露损害脑学习、记忆功能的神经机制提供资料。
材料与方法
1、动物分组与染毒
健康Wistar大鼠45只,体重250 g左右,雌雄2∶1。适应饲养环境1 w后,随机分为3组:对照组、低剂量组和高剂量组,每组15只(雌雄2∶1)。对照组:饮用蒸馏水;低剂量组:饮用含0.2%AlCl_3的蒸馏水溶液(用0.2%-Al表示);高剂量组:饮用含0.4%AlCl_3的蒸馏水溶液(用0.4%-Al表示)。各组自染毒始即分别合笼,子代鼠出生后通过母乳继续染毒,断乳后(生后21天)各组子代鼠对应分别以蒸馏水或母鼠染毒剂量的AlCl_3溶液喂养,至子鼠生后90天。然后,按原母鼠染毒的剂量分组,每窝取1～2只子鼠(雌雄各半),组成子代各实验组,以蒸馏水喂养。动物室温度18℃～23℃,相对湿度45～55%。
2、脑组织及血液中铝含量测定
准确称取0.1～0.5 g的脑组织或准确吸取0.2～0.5 ml的全血于石英烧杯中,加5～8 ml混酸,同时做空白对照。采用原子吸收石墨炉法测定脑铝和血铝含量。
3、学习和记忆的行为学测定
测试方法为跳台法(即步下试验,Step-down)。学习行为训练:将大鼠放入跳台仪内适应3 min后训练其找到平台三次,大鼠跳下平台时,立即通电给予持续电刺激(36 V)并开始计时,记录5 min内大鼠受到电刺激第一次跳上平台躲避电击的反应时间(Escape latency,EL)以及受到电击的次数(Number of errors)。24 h后进行记忆保持测试:记录大鼠置于平台到第1次跳下平台的时间(Step-downlatency,SDL)和5 min内受到电击的次数(错误次数)。
4、电生理LTP测定
乌拉坦麻醉后,将动物头部固定于脑立体定位仪上,在Schaffer侧枝部位(坐标:前囟后3.3 mm;旁开3.8 mm;皮层下3.8 mm)插入刺激电极。将记录玻璃微电极插入海马CA1区部位(坐标:前囟后3.3 mm;旁开1.5 mm;电极尖端先抵皮层表面,然后逐步推进)行细胞外记录。找到稳定的群体锋电位(populationspike,PS)后,首先记录30 min,每分钟给予一次单脉冲刺激所诱发的PS。然后观察给予同样强度及波宽的的短串高频条件刺激后,每分钟给予一次单脉冲检验刺激所诱发的PS的幅值变化。
5、海马细胞内游离钙的测定
心脏采血并取脑,分离出海马组织并去除脑膜血管,制备成单细胞悬液,然后Fura-2/AM避光孵育负载,调激发波长340 nm,发射波长510 nm,采用日立F-3000型荧光双波长分光光度计测定。
6、海马钙调蛋白依赖性激酶Ⅱ(CaMKⅡ)的表达
实验后大鼠立即断头取脑分离并取双侧海马组织后按体积比为1:6-1:9放到预冷的裂解缓冲液中。4℃超声粉碎后离心30min,取上清分装。按照常规Westernblot方法分离蛋白质。将蛋白印迹显影图扫描,再利用ChemiImager 5500 V 2103图像分析软件对实验结果(测定目标带单位密度)进行分析。
7、统计学处理
实验所得数据资料用(?)+s表示,组间资料统计用单因素方差分析(ANOVA)检验,行为学数据不服从正态分布,采用秩和检验。
实验结果
1、各组大鼠血铝和脑铝含量的比较
随着铝暴露剂量的升高,铝暴露组大鼠的血铝、脑铝含量逐渐升高,且均显著高于对照组(P＜0.05 or P＜0.01),且两铝暴露组间差异也显著(P＜0.05)。
2、大鼠的学习记忆能力比较
与对照组相比,学习行为表现为第一次跳上平台的时间(反应时间)均明显长(P＜0.01),5 min内受到电击次数显著增多(P＜0.01)。记忆行为则表现为第一次跳下平台的潜伏期明显缩短(P＜0.01),5 min内错误次数明显增加(P＜0.01),且两铝暴露组间亦有显著性差异(P＜0.01)。
3、PS幅值的变化情况
随着铝暴露剂量的升高,高频刺激后PS幅值增强率依次降低,两铝暴露组与对照组相比,显著降低(P＜0.01),但两暴露组之间差异不显著(P＞0.05)。
4、海马神经元的细胞内[Ca~(2+)]i比较
随着铝暴露剂量的增加,海马神经元的细胞内Ca~(2+)浓度([Ca~(2+)]_i)逐渐降低。两铝暴露组与对照组相比均降低,0.2%-Al组与对照组的差异具有显著意义(P＜0.05),0.4%-Al组与对照组的差异则具有显著意义(P＜0.01),且0.2%-Al组与0.4%-Al组之间的差异也具有显著意义(＜0.05)。
5、海马CaMKⅡ的表达
与对照组相比,两铝暴露组海马CaMKⅡ的表达明显降低,具有统计学意义(P＜0.01),并且0.2%-Al组和0.4%-Al组之间亦有显著性差异(P＜0.01)。
讨论
出生前后(孕期、哺乳期、断乳后发育期)慢性铝暴露的年轻大鼠,与对照组相比其血铝和脑铝含量明显增高,LTP的增强率明显下降,学习行为表现为第一次跳上平台的时间(反应时间)均明显长,5 min内受到电击次数显著增多。记忆行为则表现为第一次跳下平台的潜伏期明显缩短,5 min内错误次数明显增加,表明母体的铝暴露可通过胎盘和血脑屏障进入子代鼠体内,造成铝在子代体内蓄积,特别是脑内的蓄积,进而损害了子代鼠的学习和记忆能力。本实验同时观察到,各组海马神经元[Ca~(2+)]_i和CaMKⅡ的表达也降低,提示出生前后连续慢性铝暴露年轻大鼠学习记忆能力下降的可能原因之一是铝干扰了海马神经元细胞内Ca~(2+)稳态平衡并进一步抑制了CaMKⅡ的表达。
有研究表明,铝可阻断电压依赖性钙通道(voltage-dependent calcium channels,VDCCs);和抑制磷脂酶C(phospholipase C,PLC)催化的磷脂酰二磷酸肌醇(phosphatidylinositol-4,5-diphosphate,PIP_2)的水解,造成作为第二信使的三磷酸肌醇(IP_3)和二酰甘油(DG)生成减少,进而影响了Ca~(2+)的内流和内质网钙库中Ca~(2+)的释放。
铝一方面降低了细胞内Ca~(2+)浓度,另一方面铝与钙竞争,抑制了钙与CaM(钙调蛋白)结合性,并使其构型改变,阻断了Ca~(2+)依赖性CaMKⅡ的作用,干扰了磷酸化过程。同时铝对CaMKⅡ参与LTP诱导与维持过程中所需的NMDA受体和AMPA受体等也有影响,因而进一步抑制了CaMKⅡ的表达。
综上所述,有理由认为,铝影响学习记忆能力的可能原因之一是其可通过多种途径引起细胞内Ca~(2+)浓度降低,并进一步抑制了CaMKⅡ的表达。
结论
(1)母体期和断乳后发育期慢性铝暴露的大鼠,与对照组相比,PS幅值增强率降低,提示该暴露损害了大鼠LTP的诱导与维持;
(2)母体期和断乳后发育期慢性铝暴露的年轻大鼠与对照组相比,学习行为表现为第一次跳上平台的时间(反应时间)均明显延长,5 min内受到电击次数显著增多。记忆行为则表现为第一次跳下平台的潜伏期明显缩短,5 min内错误次数明显增加,提示铝暴露损害了大鼠的学习和记忆行为;
(3)铝损害LTP和对神经元细胞的损伤的可能机制之一是铝造成了海马中的细胞内Ca~(2+)浓度降低;
(4)母体期和断乳后发育期慢性铝暴露可使大鼠神经元细胞内CaMKⅡ的表达受到抑制。
Introduction
Aluminum is one of the most abundant element on earth.It has neurotoxicity if much of that accumulate in human's body.Evidence of epidemiology has shown that Al can impair neuron and induce learning and memory deficiency.In experimentally induced dementia animal models,it has been demonstrated that there are a reduced capacity of learning and memory and the pathological change which is similar with Alzheimer's disease after Al exposure.
The hippocampus is the most important encephalic region relative to function of learning and memory.Hippocampal long-term potentiation(LTP)is NMDA receptor-dependent persistent enhancement of efficacy in synaptic transmission,it is reputed that LTP represents the most intensively studied synaptic model and cellular basis of learning and memory in the mammalian brain.So investigating the effect of aluminum exposure on LTP and the biochemical indicators relative to the LTP synaptic mechanism will help to elucidate the mechanism of aluminum damaging the learning and memory on synaptic and proteinic level.
Although Al has been reported to impair LTP following administration in vivo and in vitro,the underlying mechanisms of Al action on LTP are still unknown.The stage of maternal and postweaning is a very important period during brain development. When the brain is exoposed to Al during this period,whether its development especially the intelligence and learning abilities will be affected,which is an important matters concerned with human's health.There is little reports about the effect of Al on learning and memory and LTP of this stage,so it is necessary for us to have a deep research.
The present study has observed the effect of aluminum exposure during maternal and postweaning period on the offspring's learning and memory and the Ca~(2+), CaMKⅡof hippocampus,hope to elucidate the synaptic mechanisms of the impairs of Al exposure on the learning and memory.
Materials and methods
1.Group and exposure
Healthy adult Wistar rats(280-320)g were exposed to aluminum through drinking 0%(distilled water)or the concentration of Al~(3+)were 0.2 g/100ml(2 g·L~(-1))(represented by 0.2%-Al)and 0.4g/100ml(4g·L~(-1))(represented by 0.2%-Al)aluminum chloride(AlCl_3) solution,respectively,from duration of pregnancy to postnatal 90 days.Their offspring were distributed into three experimental groups:a controls group;two exposed groups (0.2%-Al group and 0.4%-Al group).And the room temperature is 18℃～23℃,relative humidity is 45%～55%.
2.Brain and blood Al determinations
The brain and blood aluminum concentrations were determined by atomic absorption plumbago.Weigh 0.1～0.5g brain tissue or take suction 0.2～0.5ml whole blood,add 5～8ml violet acid,blank together.
3.Learning and memory behavioral testing
The step-down test was used to monitor the learning and memory ability.Learning ability test:put the rat into the jumping fiat instrutment to accommodate for 3 minuts, then train them to find the flat three times,when rat jump off the flat,give them a electric stroke(36 V)and begin to record the time,recording the escape latency and the number of errors in 5 min.24 hours later,memory ability test:recording the step-down latency and the number of errors in 5min.
4.Electrophysiological recordings
The extracellular micropipette recording technique was used to recording electrophysiological change.Wistar rats were anaesthetized with 20%urethane(6.5 ml/kg,i.p.)and their heads were fixed in a stereotaxis instrument.Population spike(PS) was evoked by electrical stimulation of Schaffer Collateral pathway form the CA3 to the CA1 region of hippocampus using a concentric bipolar stimulating electrode located at the area CA3 of Schaffer collateral(3.8mm posterior to bregma,3.8mm lateral to the midline,3.8mm under cortex).Glass micropipettes filled with 3 M KCl were placed at the CA1 region of hippocampus(3.3mm posterior to bregma;1.5mm lateral to the midline;on the surface of cortex)and lowered into the area CA1 for recording filed potentials.After the response had stabililized in each rat,30 min baseline was recorded.Stimuli were given every minute(all responses were averaged for each date point).A high frequency stimulation(HFS)(100 Hz,5s)was delivered at same intensity.Testing with single pulses was continued after the HFS was delieved.
5.Intracellular Ca2+ concentration in hippocampus
Intracellular Ca~(2+)concentration in hippocampal cells was measured by the technique of Fura-2/AM calcium ions fluorescence indicator in SPF.The excitation wave is 340nm and the emission wave is 510nm.
6.The expression of CaMKⅡin hippocampus
Dislodged the hippocampus into cold lysate by 1:6-1:9 of volume ratio.Pulverized by transonic wave at 4℃and centrifuged in 12000 g 1 hour,then separated the supernatant.According to the technique of ChemiImager 5500 V 2103 picture analysis software,measured the expression of CaMKⅡin hippocampus.
7.statistical analysis
Experimental values were indicated as the mean±SD.Interclass data were tested by ANOVA.The results of behavioral testing are not normal distribution so by rank sum test.
Results
1.The comparison of blood and brain aluminum
The aluminum concentration in blood and brain tissue was significantly higher than the control group(P＜0.05 or P＜0.01)and increased with exposure dose. Furthermore,the difference between the two exposed group was statistically significant.
2.The comparison of learning and memory behavioural test
The behavioural data showed:the escape latency was remarkably prolonged,the step-down latency was remarkably decreased,and the number of errors both in learning and memory was significantly increased in the Al~(3+)exposed groups(P＜0.01 or P＜0.05) as compared to the control group.Furthermore,the difference between the two exposed group was statistically significant.
3.The comparison of electrophysiological recording results
With the increase of exposure dose,the PS magnitude reduced evidently aider HFS. As compared to the control group,the two exposed groups were significant reduced, but the difference between the two exposed groups was statistically non-significant.
4.The comparison of intracellular calcium of hippocampal cells
The hippocampus intracellular Ca~(2+)concentration was reduced in 0.2%-Al group as well as 0.4%-Al group,this reduction in 0.2%-Al group was statistically significant (P＜0.05),but was significantly decreased in 0.4%-Al exposed group(P＜0.01).The difference between 0.2%-Al and 0.4%-Al group was significantly(P＜0.05).
5.The expression of CaMKⅡin hippocampus
Compared to the control group,the expression of CaMKⅡin hippocampus were significantly reduced in 0.2%-Al group as well as 0.4%-Al group(P＜0.01).
Discussion
The young rats of Al exposure from prenatal to postnatal(maternal,pregnancy and postweaning)period could increase the level of Al in blood and brain,evidently decrease the amplitude of PS afer HFS,prolong remarkably the escape latency, shortened the latent period of jumping off the flat,increase the erring times during the 5 minutes in learning and memory behavioural test,it express that the maternal and postweaning period of Al exposure impaired cognitive function of the offspring through placenta and blood brain barrierand resulted in the accumulate of Al in thebrain of the offspring,impaired the offspring's learningand memory ability.In this study,the result also showed the decrease of intracellular Ca~(2+)and the expression of CaMKⅡin hippocampus,this suggested one of the reasons that maternal and postweaning chronic aluminum impaired the offspring's learning and memory ability is disturbing the intracellular Ca~(2+),balance,and further inhabit the expression of the CaMKⅡ.
The present results also showed that maternal and postweaning chronic Al exposure could decrease the intracellular Ca~(2+)concentration in hippocampus,which was dose-dependent.Some reports showed that Al meight interfere with[Ca~(2+)]_i by block the VDCCs;inhibited the expression of NMDA receptor in hippocampus;IP_3 decreaseaffected the Ca~(2+)inflowing and degraded the intracellular Ca~(2+)release from calcium reservoir pool.
In one aspect that Al decreased intracellular Ca~(2+)concentration,another Al competed with Ca~(2+)to integrate with CaM,moreover,altered it's configuration then interrupted the contribution of Ca~(2+)/CaMKⅡ,interfered with phosphorylation cours.At same time Al also affected NMDA receptor and AMPA receptor,consequently inhibited the expression of CaMKⅡ.
In a word,Al affected Ca~(2+)concentration and the expression of CaMKⅡin hippocampus by many means which played an important role in LTP.It's needed to be further investigations.
Conclusion
1.Chronic Al exposure from prenatal to postnatal period can impair the behaviour of the learning and memory in young rats;
2.Chronic Al exposure from prenatal to postnatal can make the amplitude of PS decrease,impaired the induction and maintenance of LTP in young rats.
3.One of the probable mechanisms of impairing LTP may be the decrease of [Ca~(2+)]i in hippocampus.
4.Another probable mechanisms of impairing LTP may be the decrease of CaMKⅡexpression by Al.
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37 Silva AJ,Paylor R,Wehner JM,et al.Imapalred spatial learning in alpha calcium calmodulin dinase Ⅱ mrtant mice.Science.1992;257(5067):206-211.
38 Bayer KU,De Koninck P,Leonard AS et al.Interaction with the NMDA receptor locks CaMK in an active conformation.Nature.2001;411:801-805.
39 Yang Shi and Iryna M.Ethell.Integrins Control Dendritic Spine Plasticity in Hippocampal Neurons through NMDA Receptor andCa2+/Calmodulin-Dependent Protein Kinase Ⅱ-Mediated Actin Reorganization.Neuroscience.2006;26(6):1813-1822.
40 Victor J Johnson, Sang-Hyun Kim and Raghubir P Sharma. Aluminum-Maltolate Induces Apoptosis and Necrosis in Neuro-2a Cells: Potential Role for p53 Signaling. Toxicological Sciences. 2005; 83:329-339.
41 John M Schmitt, Eric S Guire, Takeo Saneyoshi, et al. Soderling. Calmodulin- Dependent Kinase Kinase/Calmodulin Kinase I Activity Gates Extracellular- Regulated Kinase-Dependent Long-TermPotentiation. Neuroscience. 2005; 25(5): 1281-1290.
1 Bliss TVP,Lomo T.Long-lastting potentiation of synaptic transmission in the dentate area of anaesthetized rabbit following stimulation of the perforant path[J].J Physiol,1973,232:331-356.
2 Bliss TVP,Collingridge GL.A synaptic model of memory:long-term potentiation in the hippocampus[J].Nature,1993,361(7):31-39.
3 Greenough WT,Juraska JM,Volkmar FR.Maze training effects on dendritic branching in occipital cortex of adult rats[J].Behav Neural Biol,1979,26(3):287-297
4 lisman je.A mechanism for memory storage insensitive to molecular turnover:a bistable autophosphorylating kinase[J].Proc Natl Acad Sci U S A,1985,82(9):3055-7
5 Saitoh T,Schwartz JH.Phosphorylation-dependent subcellular translocation of a Ca2+/calmodulin-ependent protein kinase produces an autonomous enzyme in Aplysia neurons[J].J Cell Biol,1985,100(3):835-842
6 Sanna PP.Cammaller M.Berton F,et al.Phosphatidylinositol 3-kinase is required for the expression but not for the induction or the maintenance of long-term potentiation in the hippocampal CAl region.J Neurosci,2002;22(8):3359-3365.
7 Silva AJ,Rosahl Tw,Chapman PF,et al.Impaired learning in mice with abnormal short-lived plasticity.Curr Biol,1996;6:1509-1518
8 蔡文琴.医学神经生物学基础.重庆:西南师范大学出版社。2001:355
9 Byzov AL.Efficacy of synaptic transmission controlled by morphology of photoreceptors of vertebrates and eletroreceptors of the ampullase of lorenzin.Sensory Systems,1994;8:3-4
10 K Fukunaga,L Stoppini,E Miyamoto,and D Muller;Long-term potentiation is associated with an increased activity of Ca2+/calmodulin-dependent protein kinase Ⅱ;J.Biol.Chem.,Apr 1993;268:7863-7867
11 Silva AJ,Paylor R et al.Imparied spaftal learning inalpha-calcium-calmodvlin kinase Ⅱ mufant mice[J].Science,1992,257:206-211
12 Pettit DL,Perlmam S,Malinow R.Potentiated transmission and prevention of further LTP by increased CaMKⅡ activity in postsynaptic hippocampal slice neurons[J].Science,2004,266,1881-1885
13 Gies KP,Fedorov NB et al.Autophosphorylation at Thr286 of the α Calcium- Calmodulin Kinase Ⅱ in LTP and Learning[J].Science,1998,279,870-873
14 Mayford M,Bach ME,et al.Control of Memory Formation Through Regulated Expression of a CaMKⅡ Transgene[J].Science,2003,274,1678-1683
15 Bach ME,Hawkins RD et al.Impairment of spatial but not contextual memory in CaMKⅡmutant mice with a selective loss of hippocarnpal LTP in the range of the θ frequency[J].Cell,1995,81,905-915
16 Stefan Strack and Roger J.Colbran;Autophosphorylation-dependent Targeting of Calcium/Calmodulin-dependent Protein Kinase Ⅱ by the NR2B Subunit of the N-Methyl-D-aspartate Receptor:J.Biol.Chem.,Aug 1998;273:20689-20692;
17 W.Muller,J.J.Petrozzino,L.C.Griffith,W.Danho,and J.A.Connor;Specific involvement of Ca(2+)-calmodulin kinase Ⅱ in cholinergic modulation of neuronal responsiveness;J Neurophysiol,Dec 1992;68:2264-2269.
18 唐开福,张银辉,袁建刚.CaMKⅡ在学习与记忆中的作用[J].《生物工程进展》,2001,21(2):50-53
19 Joiner,M.A.and L.C.Griffith.CaM kinase Ⅱ and visual input modulate memory formation in the neuronal circuit controlling courtship conditioning[J].Neurosci.1997,17:9384-9391
20 Fan GH,Wang Lz et al.Inhibition of calciumoelulin-dependent protein kinase Ⅱ in rat hippocampus attenuates morphine tolerance and dependence[J].Mol pharmacol,1999,56:39-45
21 Malenka RC,Malenka RC,Kauer JA,et al.An essential role for postsynaptic calmodulin and protein kinase activity in long- term potentiation[J].Nature,1989,340(6):554-557.
22 John EL,Justin RF.What Maintains Memories?[J].Science,1999,283(15):339-340.
23 Barria A,Muller D.Regulatory phosphorylation of AMPA-type Glutamate receptors by CaMK Ⅱ during long-term potentiation[J].Science,1997,276(9):2042-2043.
24 Karl PG,Nikolai BF,Robert KF,et al.Autophosphorylation at Thr 286 of the calciumcalmodulin kinase Ⅱ in LTP and Learning[J].Science,1998,279(7):870-873.
25 Shen K,Meyer T.Dynamic control of CaMK Ⅱ translation and localization in hippocampal neurons[J]. Science ,1999,284 (3):162 -167.
26 Meyer T, Hanson PI, Schulman H. Calmodulin trapping by calcium/ calmodulin - dependent protein kinase[J].Science ,1992,256(11):1199 -1202.
2 PerlD P,Gajdusck C D,Garruto R M et al.Intraneuronal aluminum accumulation in amyotrophic lateral sclerosis and Parkinsonism-dementia of Guam.Science,1982;217:1053.
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28 沈丽,张琳,南燕.NMDA受体与中枢神经系统发育.生理科学进展,1999;30(1):60-62.
29 Silva AJ,Rosahl Tw,Chapman PF,et al.Impaired learning in mice with abnormal short-lived plasticity.Curr Biol,2005;6:1509-1518.
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33 T Torliska,A Grochowalska.Age-realated changes of Na+,K+- ATPase,Ca2+- ATPase and Mg2+-ATPase activities in rat brain synaptosomes.Physiology and Pharmacology.2004;55(2):457-465.
34 Fang-Min Lu and Robert D.Hawkins.Presynaptie and postsynaptic Ca2+ and CaMKⅡcontribute to long-term potentiation at synapses between individual CA3 neurons.PNAS.2006;11(103):4264-4269.
35 Soderling TR,Chang B,Brickey D.Cellular signaling through multifunctional Ca2+/calmodul in-dependent protein kinase Ⅱ.Journal of Biological Chemistry,2001;276(6)3719-3722.
36 Charles CF,Tobias M.Molecular mechanisms of CaMK Ⅱ activation in neuronal plasticity.Curr Opin in Neurobiol,2002;12(2):293-9.
37 Silva AJ,Paylor R,Wehner JM,et al.Imapalred spatial learning in alpha calcium calmodulin dinase Ⅱ mrtant mice.Science.1992;257(5067):206-211.
38 Bayer KU,De Koninck P,Leonard AS et al.Interaction with the NMDA receptor locks CaMK in an active conformation.Nature.2001;411:801-805.
39 Yang Shi and Iryna M.Ethell.Integrins Control Dendritic Spine Plasticity in Hippocampal Neurons through NMDA Receptor andCa2+/Calmodulin-Dependent Protein Kinase Ⅱ-Mediated Actin Reorganization.Neuroscience.2006;26(6):1813-1822.
40 Victor J Johnson, Sang-Hyun Kim and Raghubir P Sharma. Aluminum-Maltolate Induces Apoptosis and Necrosis in Neuro-2a Cells: Potential Role for p53 Signaling. Toxicological Sciences. 2005; 83:329-339.
41 John M Schmitt, Eric S Guire, Takeo Saneyoshi, et al. Soderling. Calmodulin- Dependent Kinase Kinase/Calmodulin Kinase I Activity Gates Extracellular- Regulated Kinase-Dependent Long-TermPotentiation. Neuroscience. 2005; 25(5): 1281-1290.
1 Bliss TVP,Lomo T.Long-lastting potentiation of synaptic transmission in the dentate area of anaesthetized rabbit following stimulation of the perforant path[J].J Physiol,1973,232:331-356.
2 Bliss TVP,Collingridge GL.A synaptic model of memory:long-term potentiation in the hippocampus[J].Nature,1993,361(7):31-39.
3 Greenough WT,Juraska JM,Volkmar FR.Maze training effects on dendritic branching in occipital cortex of adult rats[J].Behav Neural Biol,1979,26(3):287-297
4 lisman je.A mechanism for memory storage insensitive to molecular turnover:a bistable autophosphorylating kinase[J].Proc Natl Acad Sci U S A,1985,82(9):3055-7
5 Saitoh T,Schwartz JH.Phosphorylation-dependent subcellular translocation of a Ca2+/calmodulin-ependent protein kinase produces an autonomous enzyme in Aplysia neurons[J].J Cell Biol,1985,100(3):835-842
6 Sanna PP.Cammaller M.Berton F,et al.Phosphatidylinositol 3-kinase is required for the expression but not for the induction or the maintenance of long-term potentiation in the hippocampal CAl region.J Neurosci,2002;22(8):3359-3365.
7 Silva AJ,Rosahl Tw,Chapman PF,et al.Impaired learning in mice with abnormal short-lived plasticity.Curr Biol,1996;6:1509-1518
8 蔡文琴.医学神经生物学基础.重庆:西南师范大学出版社。2001:355
9 Byzov AL.Efficacy of synaptic transmission controlled by morphology of photoreceptors of vertebrates and eletroreceptors of the ampullase of lorenzin.Sensory Systems,1994;8:3-4
10 K Fukunaga,L Stoppini,E Miyamoto,and D Muller;Long-term potentiation is associated with an increased activity of Ca2+/calmodulin-dependent protein kinase Ⅱ;J.Biol.Chem.,Apr 1993;268:7863-7867
11 Silva AJ,Paylor R et al.Imparied spaftal learning inalpha-calcium-calmodvlin kinase Ⅱ mufant mice[J].Science,1992,257:206-211
12 Pettit DL,Perlmam S,Malinow R.Potentiated transmission and prevention of further LTP by increased CaMKⅡ activity in postsynaptic hippocampal slice neurons[J].Science,2004,266,1881-1885
13 Gies KP,Fedorov NB et al.Autophosphorylation at Thr286 of the α Calcium- Calmodulin Kinase Ⅱ in LTP and Learning[J].Science,1998,279,870-873
14 Mayford M,Bach ME,et al.Control of Memory Formation Through Regulated Expression of a CaMKⅡ Transgene[J].Science,2003,274,1678-1683
15 Bach ME,Hawkins RD et al.Impairment of spatial but not contextual memory in CaMKⅡmutant mice with a selective loss of hippocarnpal LTP in the range of the θ frequency[J].Cell,1995,81,905-915
16 Stefan Strack and Roger J.Colbran;Autophosphorylation-dependent Targeting of Calcium/Calmodulin-dependent Protein Kinase Ⅱ by the NR2B Subunit of the N-Methyl-D-aspartate Receptor:J.Biol.Chem.,Aug 1998;273:20689-20692;
17 W.Muller,J.J.Petrozzino,L.C.Griffith,W.Danho,and J.A.Connor;Specific involvement of Ca(2+)-calmodulin kinase Ⅱ in cholinergic modulation of neuronal responsiveness;J Neurophysiol,Dec 1992;68:2264-2269.
18 唐开福,张银辉,袁建刚.CaMKⅡ在学习与记忆中的作用[J].《生物工程进展》,2001,21(2):50-53
19 Joiner,M.A.and L.C.Griffith.CaM kinase Ⅱ and visual input modulate memory formation in the neuronal circuit controlling courtship conditioning[J].Neurosci.1997,17:9384-9391
20 Fan GH,Wang Lz et al.Inhibition of calciumoelulin-dependent protein kinase Ⅱ in rat hippocampus attenuates morphine tolerance and dependence[J].Mol pharmacol,1999,56:39-45
21 Malenka RC,Malenka RC,Kauer JA,et al.An essential role for postsynaptic calmodulin and protein kinase activity in long- term potentiation[J].Nature,1989,340(6):554-557.
22 John EL,Justin RF.What Maintains Memories?[J].Science,1999,283(15):339-340.
23 Barria A,Muller D.Regulatory phosphorylation of AMPA-type Glutamate receptors by CaMK Ⅱ during long-term potentiation[J].Science,1997,276(9):2042-2043.
24 Karl PG,Nikolai BF,Robert KF,et al.Autophosphorylation at Thr 286 of the calciumcalmodulin kinase Ⅱ in LTP and Learning[J].Science,1998,279(7):870-873.
25 Shen K,Meyer T.Dynamic control of CaMK Ⅱ translation and localization in hippocampal neurons[J]. Science ,1999,284 (3):162 -167.
26 Meyer T, Hanson PI, Schulman H. Calmodulin trapping by calcium/ calmodulin - dependent protein kinase[J].Science ,1992,256(11):1199 -1202.