Quantitative analysis of experimental animals
Experiment data were obtained in all of the 15 rats for result analysis. No rats were off.

Post-high-frequency stimulation long-term potentiation change in the CA1 field of rat right hippocampus

In the control group, long-term potentiation was successfully induced, mean (144.0±16.0)% (n=5), maximum 172.3%, appeared at about 2.0-2.5 hours after high-frequency stimulation; for the amyloid beta-protein treated rats, long-term potentiation was significantly inhibited, mean (99.5±4.7)% (n=5, P < 0.001); in the integrin beta-1 subunit inhibitor group, long-term potentiation presented obviously recovery, which was close to the control, mean (141.9±7.4)% (n=5, P > 0.05), maximum 159.2%, appeared at 2-2.9 hours post- high-frequency stimulation (Figure 1).

Post-high-frequency stimulation excitatory postsynaptic potential change in the CA1 field of rat right hippocampus (Table 1)

It was found that the peak in the control group occurred after 2 hours (P < 0.01); for the amyloid beta-protein treated rats, long-term potentiation was consistently flat after high-frequency stimulation without any remarkable potentiation (P > 0.05); in the integrin beta-1 subunit inhibitor group, the peak was also at 2 hours post-high-frequency stimulation (P < 0.01; Figure 2).

[1] Blennow K, de Leon MJ, Zetterberg H. Alzheimer's disease. Lancet. 2006;368(9533):387-403. [2] Lee HG, Moreira PI, Zhu X, et al. Staying connected: synapses in Alzheimer disease. Am J Pathol. 2004;165(5): 1461-1464. [3] Doherty GH, Beccano-Kelly D, Yan SD, et al. Leptin prevents hippocampal synaptic disruption and neuronal cell death induced by amyloid β. Neurobiol Aging. 2013;34(1): 226-237. [4] Tyan SH, Shih AY, Walsh JJ, et al. Amyloid precursor protein (APP) regulates synaptic structure and function. Mol Cell Neurosci. 2012;51(1-2):43-52. [5] Chapman TR, Barrientos RM, Ahrendsen JT, et al. Synaptic correlates of increased cognitive vulnerability with aging: peripheral immune challenge and aging interact to disrupt theta-burst late-phase long-term potentiation in hippocampal area CA1. J Neurosci. 2010;30(22): 7598-7603. [6] Hamilton A, Holscher C. The effect of ageing on neurogenesis and oxidative stress in the APP(swe)/PS1(deltaE9) mouse model of Alzheimer's disease. Brain Res. 2012;1449:83-93. [7] Lisman J, Spruston N. Postsynaptic depolarization requirements for LTP and LTD: a critique of spike timing-dependent plasticity. Nat Neurosci. 2005;8(7): 839-841. [8] Malenka RC, Bear MF. LTP and LTD: an embarrassment of riches. Neuron. 2004;44(1):5-21. [9] Puzzo D, Arancio O. Fibrillar beta-amyloid impairs the late phase of long term potentiation. Curr Alzheimer Res. 2006; 3(3):179-183. [10] Townsend M, Shankar GM, Mehta T, et al. Effects of secreted oligomers of amyloid beta-protein on hippocampal synaptic plasticity: a potent role for trimers. J Physiol. 2006; 572(Pt 2):477-492. [11] Scopes DI, O’Hare E, Jeggo R, et al. Aβ oligomer toxicity inhibitor protects memory in models of synaptic toxicity. Br J Pharmacol. 2012;167(2):383-392. [12] Klein WL. Synaptotoxic amyloid-β oligomers: a molecular basis for the cause, diagnosis, and treatment of Alzheimer’s disease? J Alzheimers Dis. in press. [13] Giancotti FG, Ruoslahti E. Integrin signaling. Science. 1999;285(5430):1028-1032. [14] Chavis P, Westbrook G. Integrins mediate functional pre- and postsynaptic maturation at a hippocampal synapse. Nature. 2001;411(6835):317-321. [15] Rohrbough J, Grotewiel MS, Davis RL, et al. Integrin-mediated regulation of synaptic morphology, transmission, and plasticity. J Neurosci. 2000;20(18): 6868-6878. [16] Sabo S, Lambert MP, Kessey K, et al. Interaction of beta-amyloid peptides with integrins in a human nerve cell line. Neurosci Lett. 1995;184(1):25-28. [17] Huang Z, Shimazu K, Woo NH, et al. Distinct roles of the beta 1-class integrins at the developing and the mature hippocampal excitatory synapse. J Neurosci. 2006;26(43): 11208-11219. [18] Bernard-Trifilo JA, Kramár EA, et al. Integrin signaling cascades are operational in adult hippocampal synapses and modulate NMDA receptor physiology. J Neurochem. 2005;93(4):834-849. [19] McGeachie AB, Cingolani LA, Goda Y. Stabilising influence: integrins in regulation of synaptic plasticity. Neurosci Res. 2011;70(1):24-29. [20] Lin B, Arai AC, Lynch G, et al. Integrins regulate NMDA receptor-mediated synaptic currents. J Neurophysiol. 2003;89(5):2874-2878. [21] The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30. [22] Wang Q, Klyubin I, Wright S, et al. Alpha v integrins mediate beta-amyloid induced inhibition of long-term potentiation. Neurobiol Aging. 2008;29(10):1485-1493. [23] Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science. 2002;297(5580):353-356. [24] Anderson KL, Ferreira A. alpha1 Integrin activation: a link between beta-amyloid deposition and neuronal death in aging hippocampal neurons. J Neurosci Res. 2004;75(5): 688-697.

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Alzheimer’s disease is the most prevalent neurodegenerative disease and the most common cause of elderly dementia. Three major pathology features of Alzheimer’s disease are as follows, the extracellular abnormal deposition of amyloid beta-protein and the senile plaque formed by glial cell activation, the neurofibrillar tangles formed by intracellular tau-protein’s abnormal phosphorylation, dysfunction of synapses and neuron progressive death^[1].

Amyloid beta-protein is the major component of senile plaque, which is one of the key pathologic mechanisms of Alzheimer’s disease. It is reported that in Alzheimer’s disease, cognitive dysfunction presents earlier than the senile plaque forming. Thus it is also believed that Alzheimer’s disease is a “synaptive disorder”^[2]. Recent research has proved that accumulation of amyloid beta-protein is a key to mediating cognitive deficits in Alzheimer’s disease as amyloid beta-protein promotes synaptic dysfunction and triggers neuronal death^[3-4]. The abnormalities of synaptic structure and function are the pathology of cognitive dysfunction in the early stage of Alzheimer’s disease^[5], while senile plaques are the results in the late period^[6]. Long-term potentiation is a universally accepted electrophysiology parameter which reflects synapse plasticity^[7-8]. Lately, it is reported that the induction of long-term potentiation can be inhibited by exogenous amyloid beta-protein^[9-10]. This inhibition is recognized as the foundation of synaptic silence in the early stage of Alzheimer’s disease^[11-12].

Integrin family is a class of cellular adhesive molecules with adhesive and signal-transduction function, mediating the interaction between cell-cell and cell-extracellular matrix^[13]. Integrin is a heterodimer with α and β subunits through non-covalent binding. Subunit α has 16 subunits, while β has nine subunits. Their combination forms 24 heteromeric bodies. The subunits which have close relationship with neurosynaptic function includes α1, α3, α5, β1 and β3^[14-15]. One amino acid sequence of amyloid beta-protein, arginine-histidine-aspartate, is structurally similar with a recognition sequence of integrin, arginine-glycine-aspartate, which is possibly the binding site between amyloid beta-protein and integrin, and works as a structural basis of their combination^[16]. Existing evidence has shown that inhibition of integrin activation in mouse hippocampal slices cannot impair the induction of long-term potentiation, but can bring about its rapid decrease, which indicate integrin is critical to the maintainability and stability of long-term potentiation after induction^[17]. Another research observed that during the maturation of synapse there was reduction of glutamate releasing and transduction of N-methyl-D-aspartic acid receptor subunits, all of which could be blocked by integrin inhibitor and specific antibody of β3 receptor, thus indicating that integrin mediates the transformation of the synapse from immature to maturation^[18]. Moreover, recent studies have implicated integrins in α-amino-3-hydroxy- 5-methyl-4-isoxazolepropionic acid receptor trafficking and glycine receptor lateral diffusion, highlighting their multifaceted functions at the synapse^[19]. All of the above show that integrin participates in the regulation of neuron synapse activation, and is important to transmitters transferring, induction of long-term potentiation and maturation transduction of synapses^[20]. The present study was to assess the influence of integrin beta-1 subunit during the inhibition of long-term potentiation induced by amyloid beta-protein in the rat hippocampal CA1 field in vivo through electrophysiological technology.

Design
A case-control animal experiment
Time and setting
This study was conducted at the Department of Physiology, School of Basic Medical Sciences, Huazhong University of Science and Technology, China from January 2008 to January 2010.
Materials
Fifteen healthy clean grade male Sprague-Dawley rats, weighing 250-300 g, were used in this study. All experimental animals were provided by the Animal Center, Tongji Medical College and Tongji Hospital, Huazhong University of Science and Technology, China. All protocols of this study complied with the Guidance Suggestions for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of China^[21].
The main reagents and apparatus are introduced as follows:

Methods
Experimental animals grouping
Fifteen rats were averagely randomized into three groups: control group, amyloid beta-protein group, and integrin inhibitor group.
Alzheimer’s disease sample preparation
All rats were intraperitoneally anaesthetized by 20% urethane solution (1.0-1.5 g/kg). Body temperature was controlled at about 37.0 ℃. The rats were fixed on the stereotaxic apparatus. Stereo location was according to the position of bregma: stimulating electrode located 4.2 mm post-bregma and 3.8 mm right of midline, recording electrode located 3.4 mm post-bragma and 2.5 mm right of midline, the hole for intracerebroventricular injection located 1.0 mm post-bregma and 0.9 mm right of midline^[22]. Alzheimer’s disease models were prepared with amyloid beta-protein by the method of intracerebroventricular injection. We have looked through the reference to determine the final dose was 5 μL with a concentration of 10 μmol/L^[22]. Rats in control group were injected with 5 μL normal saline.
Integrin beta-1 subunit intervention
We injected integrin beta-1 subunit selective inhibitor (JB1A) 30 minutes before injecting amyloid beta-protein. The hole for JB1A intracerebroventricular injection located in 1.0 mm post-bregma and 0.9 mm right of midline^[22]. The dose was also 5 μL with a concentration of 10 μmol/L^[22].
Electrode recording
After injecting with normal saline, amyloid beta-protein, and specific inhibitor of integrin beta-1 subunit separately, excitatory postsynaptic potential would be recorded in the right hippocampal hemisphere, stratum radiatum in the CA1 field in response to the stimulation of the ipsilateral Schaffer collateral-commissural pathway, persistently from 10 minutes before vehicle applying to 3 hours after high-frequency stimulation. More detailed method is: gain stable excitatory postsynaptic potential by regulating the depth of electrodes; record excitatory postsynaptic potential baseline for 10 minutes as test stimulation (0.033 Hz, intensity adjusted to get a 50% excitatory postsynaptic potential maximum). Saline or JB1A were then applied (10 μL in 5 minutes, intracerebroventricular injection) and excitatory postsynaptic potential baseline was recorded for 30 minutes as the same test stimulate. Then saline or amyloid beta-protein was applied (5 μL in 5 minutes, intracerebroventricular injection) and the baseline was recorded for another 10 minutes. High-frequency stimulation was 200 Hz, with intensity adjusted to get a 75% excitatory postsynaptic potential maximum, containing 10 trains of 20 stimuli, inter-stimulus interval 5 ms, inter-train interval 2 seconds. Then the parameters were recovered to test stimulate, and excitatory postsynaptic potentials were recorded for 180 minutes. In the self-comparison, 20 excitatory postsynaptic potential values were also selected for each at post-high-frequency stimulation 1, 2, 3 hours, which were compared with the baseline.
Main outcome measures
Long-term potentiation and excitatory postsynaptic potential in the rat right hippocampus.
Statistical analysis
The experimental values were mean±SEM. SPSS 16.0 was used. Two-tailed paired t-test and non-paired t-test were used for statistical comparison. A value of P < 0.05 was considered significant.

We put forward the hypothesis according to the change of synapse activation in early pathology in Alzheimer’s disease. For the treatment limitation in the late period of Alzheimer’s disease, we hope to study the early mechanism of the change of synapse function, to clarify the mechanism of Alzheimer’s disease, to explore the early clinical interview, to delay or even reverse the disease progression, and finally to improve the patients’ prognosis and living quality.

The advanced studies have shown that amyloid beta-protein has synapse toxicity before formation of senile plaques. We also have found that because amyloid beta-protein will adhere to the cell membrane in the early period, the shape of cells will change. According to that, we confer that the combination of amyloid beta-protein and integrin will start up the downstream signal pathways and lead to synapse silence. It may be the important factors of the early mechanism of Alzheimer’s disease.

Several studies have observed that administration of exogenous amyloid beta-protein via intracerebroventricular injection could actually prominently inhibit long-term potentiation in the CA1 area of rat hippocampus, confirming amyloid beta-protein is a neurotoxin in Alzheimer’s disease^[23]. Other prior studies have shown that the neurotoxicity induced by amyloid beta-protein in the rat hippocampus could be decreased by specific inhibitors to both integrin-α1 and αV^[24]. In the present experiment, pre-treatment by specific inhibitors to integrin beta-1 subunit (JB1A) was also partly effective to prevent this long-term potentiation inhibition induced by amyloid beta-protein, remarkably increasing mean and maximum of excitatory postsynaptic potential after high-frequency stimulation. Compared with the control, however, long-term potentiation was not totally recovered after pre-treatment by JB1A, which was possibly caused by the complicated mechanism of long-term potentiation inhibition. Excitatory postsynaptic potential baseline was influenced by neither amyloid beta-protein nor JB1A, both of which were only active during induction and maintaining of long-term potentiation. It may suggest that the neurotoxicity from interaction of amyloid beta-protein and integrin took place in strengthening mechanism of memory. The efficiency of integrin beta-1 subunit specific inhibitor in preventing long-term potentiation inhibition encouraged the confidence of researching more other possibly efficacy integrin inhibitors to be used as protectors of synapses in patients with Alzheimer’s disease.

During the present research, long-term potentiation was gradually increased after high-frequency stimulation, coming to the maximum in 2 hours, and then gradually decreased, but still much higher than the baseline. Our results are not quite the same with that reported in the foreign literature because of the experiment condition, such as the change of weather, the damage of electrode, the interrupt of the environment, the difference of animal species, and the animal health status. However, we have done our best to improve it. Something that we could not avoid was the electrophysiological activation influenced by extrusion, damage, excitation and intracranial pressure change through lateral ventricle injection. We have done slowly during the injection, kept the pinhead stay several minutes after the injection, covered the puncture hole with bone wax, provided intervention agents to extend excitatory postsynaptic potential recording time, making it go back in balance completely. All we have done can reduce the impact of the experiment condition as far as possible.

Taken together, we have found integrin beta-1 subunit is an important mediate-factor in neuro-dysfunction caused by amyloid beta-protein, considering prior studies about its importance in neurodegeneration. It is suggested that agents targeting integrin beta-1 subunit may be a potential therapy to early Alzheimer’s disease. The specific mechanism needs further research. And in the future, further researches would be necessary to find what way integrin inhibitor acts in the neurotoxic progression of senile plaque forming.

1   阿尔茨海默病病理过程中，远在β淀粉样蛋白沉积形成老年斑之前，β淀粉样蛋白即可通过和整合素亚基干扰神经元突触电生理活动，影响正常递质的释放，是阿尔茨海默病早期临床症状解释的假说。文章应用在体电生理检测阐明了整合素β1亚基对活体大鼠海马CA1区中长时程增强的作用。

2   β1整合素介导活体大鼠海马CA1区β淀粉样蛋白，抑制长时程增强作用，而应用其特异性的拮抗剂或抗体可以阻断这种介导作用。

1背景介绍：

阿尔茨海默病是目前老年人常见的神经系统变性疾病，是老年人痴呆最常见的病因。其主要的病理特征包括细胞外β淀粉样蛋白异常沉积和胶质细胞活化形成老年斑、细胞内tau蛋白异常磷酸化形成神经纤维缠结、突触结构功能障碍和神经元进行性死亡。

淀粉样蛋白前体经过β和γ两种分泌酶裂解产生β淀粉样蛋白1-40（42）。老年斑是阿尔茨海默病病理机制核心因素之一，而β淀粉样蛋白则是老年斑的主要组成成分。目前已有研究表明，阿尔茨海默病远在老年斑形成之前，认知功能障碍已经表现得十分突出，因此也有观点认为，阿尔茨海默病是一种突触病。突触结构和功能的异常是阿尔茨海默病早期认知功能障碍的病理基础，而老年斑形成则是疾病相对晚期的病理结果。

长时程增强是一项公认的反映突触可塑性的电生理指标。近来有报道称它的诱导可被外源性β淀粉样蛋白抑制。长时程增强的这种抑制，被认为是阿尔茨海默病早期突触沉默的基础。

整合素家族是一族细胞粘附分子，具备粘附和信号转导功能，介导细胞之间以及细胞和细胞外基质之间的互动。它是一种由α、β亚基非共价结合的异二聚体。结构分析表明，α亚基有16个亚单位，β亚基有9个亚单位，它们的不同亚单位组合形成24种异聚体，与神经突触功能有密切联系的亚基包括：α1、α3、α5、β1和β3。Aβ的氨基酸序列RHD（精氨酸-组氨酸-天冬氨酸）在结构上与整合素的识别序列RGD（精氨酸-甘氨酸-天冬氨酸）类似，可能是β淀粉样蛋白与整合素的结合位点，是二者结合的结构基础。有研究表明，对小鼠海马脑片抑制整合素活动后虽然不会影响长时程增强的诱导，但却带来快速的长时程增强衰减，证明整合素对于诱导后长时程增强的维持和稳定起到关键的作用。一项研究发现，突触成熟过程中伴随谷氨酸释放减少和N-甲基-D-天冬氨酸受体亚单位的转换，用整合素抑制剂和β3受体特异性抗体可以阻断上述过程，证明整合素介导突触由不成熟向成熟状态的转变。综上所述，整合素参与了神经元突触活动的调控，对递质传递，长时程增强诱导以及突触的成熟转化等有重要作用。

文章研究就是关于阐明整合素的β1亚基在活体大鼠的海马CA1区β淀粉样蛋白抑制长时程增强的过程中所扮演的角色。整合素的β1亚基在β淀粉样蛋白抑制长时程增强的过程中可能起到重要的介导作用。其机制很可能在神经退行性变之前的突触功能减退中发生。

2实验过程：

实验是用雄性SD大鼠以20%乌拉坦溶液，在体温为37°C情况下，以1.0-1.5g/kg腹腔注射麻醉，NARISHIGE立体定位仪定位，根据同侧Schaffer抵押联合通路的刺激，在右侧海马记录兴奋性突触后电位。取前囟后4.2mm，右旁开3.8mm为刺激电极，前囟后3.4mm，右旁开2.5mm为记录电极，前囟后1.0mm，右旁开0.9mm为侧脑室给药孔。测试刺激频率0.033Hz，刺激强度以使电位达最大值的50%为准。高频强直刺激频率200Hz，刺激强度以使电位达最大值的75%为准，给予10组刺激，组间隔2s，波宽20ms。分别给予生理盐水，β淀粉样蛋白和β1整合素的选择性拮抗剂，记录自给予β淀粉样蛋白前10min至高频强直刺激后3h的兴奋性突触后电位。

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