【作者】 景玮；

【导师】 祁金顺；

【作者基本信息】 山西医科大学 ， 生理学， 2007， 硕士

【摘要】 阿尔茨海默病（Alzheimer’s disease,AD）是一种不可逆的原发性神经系统退行性疾病,临床表现为进行性学习、记忆和认知功能障碍,智力减退乃至完全痴呆。AD的主要病理特征有:脑内出现高密度的老年斑（senile plaque,SP）、神经原纤维缠结（neurofibrillary tangles,NFT）以及大量神经元丧失等。据分析,SP的主要成分是由39-43个氨基酸构成的淀粉样β-蛋白（Amyloidβ-protein,Aβ）。目前,有关Aβ及其有效片段如Aβ_25-35、Aβ_31-35的神经毒作用已有广泛报道,故一般认为,AD的发病与Aβ在脑内的沉积以及由此产生的神经毒性作用有关。然而,对于Aβ的神经毒性作用机制还不完全清楚,所以目前对AD的防治仍无十分有效的方法。海马是中枢神经系统中与学习和记忆关系较为密切的结构之一,也是AD时SP主要聚集的部位之一。海马的长时程增强（long term potentiation,LTP）是由强直刺激作用于海马兴奋性突触传递通路时所诱发的一种突触传递效率长时间增强的现象,被认为是一个重要的、反映信息储存过程中突触传递发生可塑性变化的电生理指标,与某种学习记忆机制有关。转基因小鼠实验证明,海马LTP的损伤与记忆行为的伤害密切相关,故海马LTP不仅被认为是突触可塑性的功能指标之一,也是研究认知功能障碍疾病如AD的理想模型。海马双脉冲易化（paired pulse facilitation,PPF）是由前后两个刺激作用于突触传递通路时引起的突触传递短时间增强现象,也被认为是反映突触可塑性的指标之一,其形成机制与突触前神经末梢递质释放的增加有关。精氨酸加压素（arginine vasopressin,AVP）是一种神经内分泌激素,作用广泛,不仅可以调节体内水电解质平衡及调节血压,存在于中枢神经系统中的AVP对学习记忆的调节过程也具有重要的意义。有研究表明AD患者脑内和血浆内AVP水平与正常人相比有明显减少,提示AD的发病可能与AVP在脑和血浆中的水平下降有关。此外AVP对老年人记忆的改善已有临床报道,提示AVP可能具有对抗Aβ神经毒性的作用,可能在防治AD、改善记忆障碍方面发挥重要作用。但迄今为止,关于AVP的实验主要集中在观察动物行为学的改变上,有关电生理学方面的机制性研究,特别是AVP对海马LTP的效应研究甚少,并且仍然存在分歧。此外,虽然有实验表明Aβ对LTP具有伤害性作用、AVP则可能与之相反,但两者合用后,AVP是否能够逆转或部分拮抗Aβ对LTP的压抑效应至今仍未见报道。因此,本实验的目的主要是进一步确定脑室内注射AVP是否能影响海马LTP,以及AVP是否可以保护神经元免受Aβ对突触传递可塑性如LTP产生的伤害作用,从而为AD的发病机制以及AD的防治提供可能的新思路。考虑到AD患者记忆力下降和痴呆的发生与海马结构的受累如严重萎缩有关,而AVP也可以通过海马发挥增强记忆的作用,本实验采用细胞外记录的方法,以在体大鼠海马Schaffer侧枝-CA1锥体细胞辐射层（Schaffer collateral/stratum radiatum）突触传递通路上诱导的场兴奋性突触后电位（fEPSPs）的幅度改变为指标,观察了经侧脑室给予不同浓度的AVP、Aβ的25-35片段(Aβ_25-35)、及同时给予两种药物时对在体条件下基础性fEPSPs、高频刺激后引发的LTP以及双脉冲引起的PPF的影响,试图证实AVP在突触传递效率方面的神经调制作用、Aβ_25-35在突触传递效率方面的神经毒性作用、以及AVP是否具有保护神经元免受Aβ_25-35引起的LTP压抑作用。结果显示:（1）在稳定记录基础性fEPSPs30min的基础上,脑室内注射5μL容积的Aβ_25-35（25nmol）或不同浓度的AVP（0.1nmol,1nmol,10nmol）后,30min内各组fEPSPs幅度与给药前相比或与对照组相比均无明显变化。（2）在对照组（n=12）,给予短串高频刺激（HFS,频率200Hz,波宽50μs,脉冲数20,重复3次,间隔30秒）后观察到LTP的出现,如以HFS前的fEPSPs幅度为100%,刺激后即刻增加到206±8.3%;HFS后30min及60min时,fEPSPs幅度仍分别保持在164±6.3%和158±7.0%。（3）HFS前30min脑室注射25nmol Aβ_25-35后,HFS引发的LTP被明显抑制,LTP值在HFS后即刻、30min和60min时分别为160±9.5%,110±8.6%及98±8.4%（n=6）,与对照组相应时间点的LTP值（206±8.3%,164±6.3%及158±7.0%,n=12）相比,差异显著（P＜0.01）。（4）脑室内同时给予不同剂量（0.1nmol、1nmol、10nmo）的AVP及25nmol Aβ_25-35后,Aβ对LTP的压抑作用有不同程度的恢复。在0.1nmol AVP+Aβ组（n=6）,HFS后即刻、HFS后30min和60min时的LTP值为168±8.5%,115±6.4%及106±7.1%,与单独给予Aβ_25-35组相应时间点LTP值（160±9.5%,110±8.6%及98±8.4%,n=6）相比,LTP有一定程度的恢复,但两者无统计学差异（P＞0.05）。在1nmol AVP+Aβ_25-35组（n=6）和10nmol AVP+Aβ_25-35组（n=6）,LTP值于HFS后即刻、30min和60min时分别为203±9.5%,164±7.8%和,151±5.6%及204±11%,185±8.7%,174±5.6%,与单独给予Aβ_25-35组相比,两组分别具有统计学差异（P＜0.01）,并且显示出一定的剂量依赖性。（5）HFS前30min脑室内单独注射3种不同浓度的AVP（0.1nmol,n=7;1nmol,n=6;10nmol,n=7）后,HFS引起的LTP值于HFS后即刻、30min和60min时分别为198±12%,170±8.4%,156±9.1%;250±12%,192±7.8%,180±9.7%及253±9.7%,197±7.1%and 192±8.2%,与对照组（206±8.3%,164±6.3%及158±7.0%,n=12）相比,0.1nmol AVP组没有显著性统计学差异,1nmol组和10nmol组引导的LTP被显著增强,均有统计学差异（P＜0.01）,但两者（1nmol组和10nmol组）相比没有显著性差异（P＞0.05）。（6）上述不同剂量的AVP、Aβ_25-35及两者合用时对双脉冲刺激引起的突触后电位的增强即双脉冲易化（PPF）均无明显影响。以上结果表明,（1）上述三种浓度的AVP及25nmol的Aβ_25-35均不影响基础的突触传递过程。（2）脑室注射1nmol和10nmol AVP可显著增强高频刺激诱发的LTP,表明AVP具有上调海马CA1区突触传递及可塑性的效应。中等浓度（1nmol）和较高浓度（10nmol）AVP对LTP的增强效应之间无明显统计学差异,加之低浓度（0.1nmol）时AVP对LTP又无显著增强作用的现象提示,AVP在脑内增强LTP效应的有效浓度范围可能较小,容易产生饱和。（3）三种浓度的AVP可以在一定程度上剂量依赖性逆转Aβ_25-35引起的LTP压抑,这说明AVP具有对抗Aβ神经毒性和维持正常突触传递及可塑性的作用,也提示脑内AVP受体可能是AD时Aβ发挥神经毒性作用的靶点之一。（4）PPF不受Aβ_25-35及AVP的影响,说明Aβ_25-35对LTP的抑制及AVP对LTP的易化作用可能主要是通过突触后机制,而不是通过减弱突触前神经递质的释放实现的。以上实验进一步确定了脑室内注射AVP对海马LTP的上调作用,首次发现了AVP可以保护神经元免受Aβ对突触传递可塑性如LTP产生的伤害作用,为阐明AD的发病机制以及为有效防治AD提供了新的思路。更多还原

【Abstract】 Alzheimer’s disease（AD）is a primary irreversible neurodegenerative disorder characterized by progressive impairment of learning,memory and other cognitive functions, ultimately developing into mental deficiency and dementia.The main pathological characteristics of AD include the high density of senile plaques（SP）in the brain,neurofibrillary tangles（NFT）and loss of neuron in the affected brain regions.The main constituent of SP is amyloidβ-peptide（Aβ）constituted by 39-43 amino acids.At present,the neurotoxicity of Aβhas been widely reported.Therefore,the deposition of Aβin the brain and its neurotoxicity have been linked to the pathogenesis of AD,and the Aβhypothesis in AD has been accepted widely. However,the possible mechanisms by which Aβimpairs neurons are now still uncertain and there is still no effective measure in the prevention and treatment of AD.Hippocampus is one of the important structures in the central nervous system（CNS）,which has been related closely to learning and memory functionally.Also,hippocampus is one of the regions where SPs accumulate in the brain of AD patients.Hippocampal Long term potentiation （LTP）is a persistent enhancement of excitatory synaptic transmission induced by preceding operations of high-frequency stimulation（HFS）.The experiments with transgenic mice which highly expressed Aβin the brain show that the age dependent memory deficits are closely related to the impaired hippocampal LTP in hippocampus,which is similar to the findings in AD patients.Thus,Hippocampus LTP has long been thought to be a suitable model for not only the study of synaptic plasticity but also the elucidation of mechanisms underlying cognitive and memory disorders,such as AD.Hippocampal paired pulse facilitation（PPF）is also an enhancement in synaptic transmission,which is produced by two continual stimulations and considered to be another parameter reflecting synaptic plasticity.The mechanism of PPF is related to the neurotransmitter release from presynaptic neurons.Arginine vasopressin（AVP）is a neuropeptide involved in the endocrine regulation of the water balance and blood pressure,also plays an important role,as a central neurotransmitter,in the regulation of some central nervous functions including learning and memory.It is reported that the concentration of AVP in the brain and the blood of the AD patients decreased significantly compared with the normal,which indicates that the incidence of the AD,to a certain extent,has a relationship with the decrease of AVP level.It is reported that AVP application can improve learning and memory in aged people,which suggests that AVP may protect neurons against neurotoxicity of Aβ,and probably,AVP plays a role in the prevention and treatment of AD and improvement of memory impairment.However,Up to now,the most experiments regarding AVP mainly focused on the observation of animal behavior.The studies on the mechanisms of AVP,especially,the effects of AVP on hippocampal LTP are rarely reported or even rather controversial.In addition,although it has been shown that Aβcan impair LTP and AVP can enhance it,but whether AVP can reverse Aβ-induced LTP suppression after co-application of AVP and Aβhas not been reported up to now.Therefore,the purposes of the present study were:（1）to further clarify the effects of AVP on rat hippocampal LTP in vivo;（2） to investigate the possible protective action of AVP on Aβ-induced LTP impairment.Since the memory impairment and the dementia happened in AD are related to the degeneration of hippocampus,and the memory enhancement by AVP is also through hippocampus,we used extracellular electrophysiological technique and recorded field excitatory postsynaptic potential（fEPSP）from the Schaffer collateral/stratum radiatum of hippocampal CA1 area.We observed the effects of different concentration of AVP,Aβfragment 25-35 (Aβ_25-35),and co-application of AVP and Aβon the in vivo baseline fEPSPs,HFS-evoked LTP and PPF,and try to confirm the neuronal regulation of AVP in synapse transmission efficiency, the neurotoxicity of Aβon the synaptic plasticity,and the neuronal protection of AVP against the impairment of Aβ_25-35on hippocampal LTP.The results showed that:（1）Intracerebroventricular（i.c.v.）injection of 25nmol Aβ_25-35（in 5μL）and different concentration of AVP（0.1nmol,1nmol,10nmol）did not affect the baseline fEPSPs during 30 min of recording period.（2）In control group（n=12）,HFS（three stimulus trains,20 test pulses in each train,200 Hz,30 s inter train interval）effectively evoked LTP.The amplitude of fEPSPs increased to 206±8.3%（setting the mean baseline amplitude of fEPSPs before HFS arbitrarily as 100%）immediately at the end of HFS,and the LTP still remained at 164±6.3%,and 158±7.0%,respectively,as examined 30 min,60 min after HFS.（3）After pretreatment（i.c.v.injection）with 5μL 25nmol Aβ_25-35for 30 min,HFS-induced LTP was inhibited obviously.As examined immediately,30 min and 60 min post-HFS,the LTP value significantly decreased to 160±9.5%,110±8.6%and 98±8.4%（n=6,P＜0.01）,respectively,from 206±8.3%,164±6.3%and 158±7.0%in control group at the same time points（n=12）.（4）After co-injection of three different concentration AVP and Aβ_25-35（25nmol）,the suppressed LTP by Aβ_25-35recovered at different degree.In 0.1nmol AVP+Aβgroup,AVP produced a little recovery for the Aβ_25-35-mediated depression of LTP,but no statistical difference.The LTP value at immediately,30 min and 60 min after HFS,was 168±8.5%,115±6.4%and 106±7.1%, respectively,compared to the control group of Aβ_25-35（160±9.5%,110±8.6%and 98±8.4%, n=6,P＞0.05）.In 1nmol AVP+Aβ_25-35group and 10nmol AVP+Aβ_25-35group,the LTP at three time points mentioned above are 203±9.5%,164±7.8%and 151±5.6%;204±11%,185±8.7%and 174±5.6%,respectively,showing significant differences compared to the group of Aβ_25-35alone （P＜0.01）and a dose-dependent effect.（5）After i.c.v,injection of three different concentration AVP（0.1nmol,n=7;1nmol,n=6;10nmol,n=7）,the HFS-induced LTP at three time points are 198±12%,170±8.4%,156±9.1%;250±12%,192±7.8%,180±9.7%and 253±9.7%,197±7.1% and 192±8.2%,respectively,with significant difference in 1nmol and 10nmol AVP groups compared to control at the same time points（P＜0.01）,but without statistical difference between this two groups and between 0.1nmol AVP and control group（P＞0.05）.（6）The drugs mentioned above including AVP,Aβ_25-35,and co-application of AVP and Aβ_25-35did not affect paired stimuli-induced PPF significantly.These results indicate that:（1）0.1-10nmol AVP and 25nmol Aβ_25-35did not affect the baseline synaptic transmission.（2）Three concentration of AVP showed a dose-dependent LTP enhancement,indicating AVP can up-regulate synaptic transmission in hippocampal CA1 region. The significant enhancement of LTP by 1nmol and 10nmol AVP but without difference between both of them,in addition to no significant increase of LTP by 0.1nmol AVP,suggesting that the effective concentration of AVP in enhancing LTP may be limited in a small range,easy to reach its saturation concentration.（3）The depression of LTP induced by Aβ_25-35can be reversed by AVP in a dose-dependent manner,suggesting that AVP can counteract the neurotoxicity of Aβ_25-35and positively modulate the synaptic plasticity.（4）The PPF was not affected by AVP and Aβ,indicate that Aβ_25-35-induced depression of LTP and AVP-induced enhancement of LTP are mainly involved in the modulation of the postsynaptic processes,not the alteration of presynaptic transmitter release.更多还原