【作者】 王晓晖；

【导师】 祁金顺；

【作者基本信息】 山西医科大学 ， 生理学， 2011， 博士

【摘要】 阿尔茨海默病（Alzheimer’s disease, AD）是一种与年龄密切相关的原发性中枢神经系统退行性疾病,主要表现为进行性认知功能障碍、学习和记忆能力丧失,晚期出现严重痴呆。AD典型的病理特征是脑内出现大量、高密度以淀粉样β蛋白（amyloidβ-protein, Aβ）为主要成分的老年斑,病变主要累及脑内与学习、记忆和认知功能有关的区域,如海马和颞叶皮层等。有关Aβ的神经毒性作用已有广泛报道。无论离体还是在体实验都显示, Aβ片段如Aβ1-40、Aβ25-35、Aβ31-35均能导致神经元结构和功能的退行性改变,例如引起培养的神经元凋亡、脑组织突触可塑性下降和空间学习记忆行为伤害等。因此,关于AD病因的“Aβ学说”已被广泛认可。Aβ发挥神经毒性作用的机制之一是诱导了细胞的钙超载,从而引发细胞内产生多种钙依赖性事件,导致细胞伤害。然而,由于Aβ神经毒性作用十分广泛、复杂,涉及到的细胞和分子机制远未被加以阐明,迄今为止,临床上仍然缺乏有效针对抗Aβ的药物。因此,积极寻找能够拮抗Aβ神经毒性、维持细胞内Ca²⁺稳态免受Aβ伤害的药物或治疗手段,已成为目前治疗和预防AD的一个关键问题。近年来一个重要的发现是,另外一种与年龄相关的退行性疾病——2型糖尿病（type 2 diabetes mellitus, T2DM）在AD的发生发展过程中起到了重要的作用。AD和T2DM在发病过程、病理变化、临床表现和预后上有着惊人的相似,甚至利用控制T2DM的手段也能干预AD,减少Aβ的产生和聚集、拮抗Aβ的神经毒性。胰高血糖素样肽1（glucagon-like peptide-1, GLP-1）是一种肠分泌的促胰岛素分泌激素,由于其不会引起正常人血糖发生改变,已成为目前治疗T2DM的最新药物。GLP-1不仅可以刺激胰岛β细胞增殖和再生、促进胰岛素分泌、抑制胰高血糖素释放,也能作用于中枢,增强学习和记忆功能。有研究表明,脑内很多部位包括海马在内都有GLP-1与其受体（GLP-1R）的表达。实验还证实,GLP-1能够降低脑内Aβ水平;能够发挥神经营养因子作用,保护神经元免受谷氨酸诱导的细胞凋亡;可以有效地阻止Aβ引起的海马长时程增强（long term potentiation,LTP）的损伤。然而,迄今为止,GLP-1的神经保护作用仍缺乏完整的在体和离体实验研究证据。GLP-1是否可以有效拮抗Aβ引起的认知功能伤害,是否能够保护海马突触传递可塑性、维持细胞内Ca²⁺稳态目前仍然不清楚,其神经保护作用的机制研究也还在起步阶段。因此,本研究采用行为学观察、电生理记录和细胞内Ca²⁺测定等手段系统探讨了GLP-1的神经保护作用及其可能机制。本研究使用了GLP-1的类似物Val⁸-GLP-1（7-36）,因为,天然的GLP-1很不稳定,在血液中的半衰期只有2-3分钟,而Val⁸-GLP-1（7-36）不仅半衰期较长,还具有更强的生物活性。研究主要分为以下几部分:（1）采用Morris水迷宫技术观察了侧脑室注射Val⁸-GLP-1（7-36）对Aβ1-40诱导的大鼠空间学习记忆能力损伤的影响。（2）采用在体大鼠海马CA1区场电位记录方法,观察了侧脑室注射Val⁸-GLP-1（7-36）是否可以拮抗Aβ1-40诱导的长持续长时程增强（L-LTP）的损伤。（3）采用大鼠脑片全细胞膜片钳（whole-cell patch clamp）方法,记录了海马CA1区锥体神经元自发微兴奋性突触后电流（miniature excitatory postsynaptic currents, mEPSCs）和自发微抑制性突触后电流（ miniature inhibitory postsynaptic currents, mIPSCs ）,观察了Val⁸-GLP-1（7-36）对Aβ1-40诱导的微突触后电流变化的影响。（4）采用激光扫描共聚焦Ca²⁺荧光成像技术,观察了Val⁸-GLP-1（7-36）是否对Aβ1-40片段引起的大鼠原代培养皮层神经元Ca²⁺紊乱具有预防和逆转作用。第一部分Val⁸-GLP-1（7-36）保护大鼠空间学习记忆免受Aβ1-40引起的损伤为阐明Val⁸-GLP-1（7-36）在脑内的神经保护作用,我们采用Morris水迷宫行为学方法观察了侧脑室注射（i.c.v.）Val⁸-GLP-1（7-36）对Aβ1-40诱导的大鼠空间学习记忆能力损伤的影响,并经HE染色观察了海马CA1区锥体神经元的形态学改变。实验选用反应灵活、无视觉和运动障碍的雄性Wistar大鼠,麻醉后在脑立体定位仪导引下,应用微量注射器将药物缓慢注入右侧侧脑室,待动物清醒并恢复两周后,进行Morris水迷宫行为学测试。测试分三部分进行:定位航行实验、空间探索实验和可见平台实验。主要观察各种药物处理对大鼠逃避潜伏期、游泳距离、大鼠处于目标象限（即平台所在象限）所占的时间百分比,以及大鼠游泳速度的影响。随后将大鼠处死取海马组织进行常规HE染色观察各组大鼠CA1区锥体神经元的病理变化。实验结果显示:（1）Aβ1-40能够显著降低大鼠的空间学习、记忆能力。侧脑室注射5 nmol Aβ1-40后,在定位航行实验（隐蔽平台测试）中,与对照组相比,从开始测试后的第2到5天,大鼠寻找平台的时间和游过的距离明显延长（P<0.01）,例如,在第5天时,Aβ1-40组大鼠逃避潜伏期和找到平台所游过的距离分别为50±5.5 s和1232.8±128.3 cm,明显长于对照组的17.8±2.9 s和458.6±63.8 （P<0.01）。在第6天的空间探索实验中,Aβ1-40注射组的大鼠处于目标象限的时间占游泳总时间的百分比为28.4±3.8%,明显短于对照组的43.5±4.8% （p<0.01）。（2）Val⁸-GLP-1（7-36）单独给予剂量依赖性增强了大鼠的空间学习和记忆能力。在定位航行实验中,低剂量（0.05 pmol）Val⁸-GLP-1（7-36）不影响大鼠逃避潜伏期和找到平台所游过的距离,与对照组相比,没有明显的统计学差异（p>0.05）;然而,0.5 pmol和5 pmol Val⁸-GLP-1（7-36）均增强了大鼠的空间学习能力,在连续5天的测试中,逃避潜伏期和找到平台所游过的距离明显小于对照组（P<0.01）,并且随着Val⁸-GLP-1（7-36）浓度的升高而显著减小（P<0.01）。如测试第5天时,0.05 pmol、0.5 pmol和5 pmol Val⁸-GLP-1（7-36）组大鼠逃避潜伏期分别为15.5±1.4 s、11.1±1.1 s和6.8±1.1 s,呈现出剂量依赖性;空间探索实验中0.05 pmol、0.5 pmol和5 pmol Val⁸-GLP-1（7-36）组大鼠处于目标象限的时间占游泳总时间的百分比分别为44.9±4.4%,48.5±4.9%和51.4±3.8%,与对照组的43.5±4.8%相比逐渐延长,尤其是0.5 pmol和5 pmol Val⁸-GLP-1（7-36）组与对照组之间具有显著性差异（P<0.01）,且随着Val⁸-GLP-1（7-36）浓度的升高而升高（P<0.01）。（3）Val⁸-GLP-1（7-36）剂量依赖性改善了Aβ1-40诱发的大鼠空间学习和记忆能力损伤。在定位航行实验中,Val⁸-GLP-1（7-36）预处理的大鼠逃避潜伏期和找到平台所游过的距离明显低于Aβ1-40单独给予组（p<0.01）,且随着Val⁸-GLP-1（7-36）浓度增加而减小,例如,测试开始后的第5天,0.05 pmol、0.5 pmol和5 pmol Val⁸-GLP-1（7-36）+Aβ1-40组大鼠逃避潜伏期分别为:30.3±3.5 s、28.4±2.5 s和18.8±2.6 s,呈现出剂量依赖性。在空间探索实验中,0.05 pmol、0.5 pmol和5 pmol Val⁸-GLP-1（7-36）预处理组的大鼠处于目标象限的时间占游泳总时间的百分比分别为40.2±4.7%, 46.4±4.6%和48.3±4.7%,明显高于Aβ1-40单独给予组（p<0.01）且随着浓度的增加而逐渐增大呈现出一定的剂量依赖性。（4）可视平台实验显示,Val⁸-GLP-1（7-36）、Aβ1-40以及二者联合应用均未影响大鼠的视力和运动能力。各组游泳速度没有明显差异（p>0.05）,基本维持于19 cm/s左右。（5）Val⁸-GLP-1（7-36）减小了Aβ1-40引起的大鼠海马CA1区锥体神经元的形态学损伤。观察发现,不同浓度的Val⁸-GLP-1（7-36）单独给予后,海马CA1区锥体神经元没有出现明显病变,而Aβ1-40单独给予则引起锥体神经元数量明显减少,密度下降,部分细胞呈现出三角形或多边形的异常形态甚至伴有核固缩的表现,即使健存的细胞也表现为排列紊乱,细胞间隙增大。与Aβ1-40单独给予组相比,在三个不同浓度的Val⁸-GLP-1（7-36）+Aβ1-40组,随着Val⁸-GLP-1（7-36）浓度的增大,Aβ1-40所致病理改变如锥体神经元密度、细胞形态和细胞排列等均明显改善。以上结果表明:侧脑室注射Aβ1-40严重损害了大鼠的空间学习和记忆能力,Val⁸-GLP-1（7-36）本身可以有效改善学习、记忆能力,也可以剂量依赖性拮抗Aβ1-40引起的学习记忆能力的损伤。本实验结果提示,脑内GLP-1的高表达或使用GLP-1类似物Val⁸-GLP-1（7-36）有可能成为预防和治疗AD等神经退行性疾病的一种新策略。第二部分:Val⁸-GLP-1（7-36）保护大鼠在体海马长持续长时程增强免受Aβ1-40引起的损伤本实验利用电生理学手段,在记录反映突触传递的场兴奋性突触后电位（field excitatory postsynaptic potential, fEPSP）基础上,观察了急性侧脑室注射Aβ1-40和Val⁸-GLP-1（7-36）以及二者联合应用对大鼠海马CA 1区在体长持续长时程增强（L-LTP）的影响,旨在探讨Val⁸-GLP-1（7-36）拮抗Aβ1-40神经毒性作用的电生理学机制。实验采用成年雄性大鼠,麻醉后将其固定在脑立体定位仪上,分别将脑室套管和绑定的同心圆双极刺激电极和单极记录电极,精确插入到侧脑室或海马刺激与记录部位。通过给予海马Schaffer侧枝测试刺激和三组高频刺激（high frequency stimulation, HFS）,在海马CA1区放射层记录基础fEPSP和HFSs引起的L-LTP。经脑室套管向侧脑室内注射Aβ1-40和Val⁸-GLP-1（7-36）,观察各种药物对基础fEPSP和L-LTP的影响。实验结果显示:（1）侧脑室注射Aβ1-40不影响基础fEPSP,但显著抑制了高频刺激引起的海马在体L-LTP。侧脑室注射5 nmol Aβ1-40后,低频刺激下连续记录3小时。发现基础fEPSP没有显著改变,注射前和注射后3小时的标准化fEPSP幅度分别为99.5±1.4%和100.2±1.11% （P>0.05）。但给予三组HFS后,fEPSP平均幅度在HFSs后1h、2h和3 h时分别为129.6±3.3%、108.1±5.4%和101.1±7.2%,明显低于对照组的153.0±4.5%, 140±4.8%和139.38±5.5% （P<0.01）,表现出明显的L-LTP抑制效应。（2）Val⁸-GLP-1（7-36）本身不影响基础fEPSP,但剂量依赖性增强了HFSs诱导的在体海马L-LTP。测试刺激条件下,0.05 pmol、0.5 pmol和5 pmol Val⁸-GLP-1（7-36）实验组大鼠在侧脑室注射前和注射后3 h的平均fEPSP幅度分别为99.5±1.4%和100.2±1.1% （P>0.05）, 100.2±2.4%和99.5±4.7% （P>0.05）, 99.9±3.32%和100.0±8.6% （P>0.05）。表明各种浓度的Val⁸-GLP-1（7-36）均不影响基础突触传递。给予HFSs后,与对照组相比,0.05 pmol Val⁸-GLP-1（7-36）组没有显著影响L-LTP（P>0.05）,但0.5 pmol和5 pmol Val⁸-GLP-1（7-36）组的L-LTP值明显高于对照组（P<0.01）,且随着Val⁸-GLP-1（7-36）浓度的增加,fEPSP幅度也逐渐增加,呈现一定的剂量依赖性,例如,在HFSs 3 h后,L-LTP值分别为152.2±2.4%和164.0±4.1%,明显高于对照组的143.2±1.5% （P<0.01）。（3）侧脑室联合应用不同浓度的Val⁸-GLP-1（7-36）剂量依赖性拮抗了Aβ1-40诱导的L-LTP损伤。HFSs后3 h时,三个不同浓度（0.05,0.5和5 pmol）的Val⁸-GLP-1（7-36）+Aβ1-40组的平均fEPSP幅度分别为124.2±4.7%、132.1±3.4%和139.0±5.3%。各组平均fEPSP幅度均明显高于Aβ1-40单独给予时的fEPSP幅度（P<0.01）,且随着Val⁸-GLP-1（7-36）浓度升高,Aβ1-40引起的L-LTP的压抑效应逐渐改善。以上结果表明,Val⁸-GLP-1（7-36）预处理可剂量依赖性拮抗Aβ1-40引起的在体大鼠海马CA1区L-LTP的抑制。Val⁸-GLP-1（7-36）对海马L-LTP的这种保护效应与Val⁸-GLP-1（7-36）在行为学上的作用保持了良好的一致性,可能部分地解释了GLP-1改善大鼠空间学习记忆功能的细胞机制。第三部分Val⁸-GLP-1（7-36）对海马CA1区锥体神经元自发性突触活动的影响突触传递是神经系统活动的最基本过程,兴奋性突触和抑制性突触活动的适当调制对大脑功能的维持至关重要。为了进一步揭示Val⁸-GLP-1（7-36）发挥神经保护作用的细胞电生理机制,本研究采用大鼠脑片全细胞膜片钳方法记录了海马CA1区锥体神经元自发性微兴奋性突触后电流（miniature excitatory postsynaptic currents, mEPSCs）和微抑制性突触后电流（miniature inhibitory postsynaptic currents, mIPSCs）,并对Val⁸-GLP-1（7-36）预处理是否干预Aβ1-40诱导的微突触后电流的变化进行了研究。主要观察指标有:mEPSCs和mIPSCs的电流幅度、发放频率、10-90%上升时间和衰减时间等。结果显示:（1）Aβ1-40显著降低mEPSCs和mIPSCs的发生频率,并延长了mIPSCs的衰减时间。100 nM Aβ1-40单独处理后,标准化mEPSCs频率较对照组降低了62.1%,由100.0±11.2%降低为37.9±8.8% （P<0.01）;标准化mIPSCs频率较对照组降低了53.5%,由100.0±5.1%降低为46.5±6.8%（P<0.01）;标准化衰减时间值为150.2±13.4%,较对照组的100.0±6.7%明显升高（P<0.01）。（2）Val⁸-GLP-1（7-36） （10 nM）没有明显改变mEPSCs和mIPSCs幅度、频率、10-90%上升时间和衰减时间（P>0.05）。（3）Val⁸-GLP-1（7-36） （10 nM）预处理拮抗了Aβ1-40 （100 nM）所致的mEPSCs和mIPSCs的频率降低和mIPSCs衰减时间的延长。在Val⁸-GLP-1（7-36）+Aβ1-40组,标准化mEPSCs频率为63.5±5.0%,明显高于Aβ1-40单独处理组（37.9±8.8%,P<0.01）;标准化mIPSCs频率为72.4±2.2%,也明显高于Aβ1-40单独处理组的46.5±6.8%（P<0.01） ;标准化mIPSCs衰减时间值为124.1±10.8%,显著低于Aβ1-40组的150.2±13.4% （P<0.01）。以上结果表明:Aβ1-40可引起mEPSCs和mIPSCs频率降低,并改变mIPSCs的通道动力学特征;Val⁸-GLP-1（7-36）预处理有助于拮抗这种变化,重新调整突触和通道活动。这些结果可能有助于解释Aβ1-40诱导的海马突触可塑性的损伤,其中自发性mEPSCs的频率下降可能主要影响突触前递质释放,而mIPSCs频率的降低有可能使神经系统兴奋和抑制的平衡遭到破坏而增强了细胞的兴奋毒作用。由此看来,Val⁸-GLP-1（7-36）对Aβ1-40诱导的mEPSCs和mIPSCs的改变实质上起到了积极的调节作用,这可能是GLP-1拮抗Aβ1-40所致神经毒性的作用机制之一,提示GLP-1以及类似物具有潜在的逆转AD退行性变的治疗作用。第三部分Val⁸-GLP-1（7-36）对海马CA1区锥体神经元自发性突触活动的影响第四部分Val⁸-GLP-1（7-36）对抗Aβ1-40引起的大鼠皮层神经元钙超载本实验采用激光扫描共聚焦显微镜成像系统进行细胞内Ca²⁺荧光成像实验,观察了Aβ1-40对原代培养的大鼠皮层神经元细胞内Ca²⁺浓度(intracellular calcium concentration, [Ca²⁺]i)的影响以及可能机制,并研究了Val⁸-GLP-1（7-36）对Aβ1-40诱导的钙稳态紊乱的保护作用。实验结果显示:（1）Aβ1-40显著增加了大鼠原代培养的大脑皮层神经元细胞内的钙水平。将培养的大鼠大脑皮层细胞用Aβ1-40（10μM）处理18分钟后,神经元的相对荧光强度从给药前的对照值（100%）明显上升到191.1±21.6% （P<0.01）;用5μM Nicardipine（特异性L-VDCC阻断剂）或50μM AP-5（特异性NMDA受体阻断剂）预处理后再用Aβ1-40干预培养细胞,均明显压抑了Aβ1-40诱导的[Ca²⁺]i的升高,相对荧光强度值分别为152.1±1.7%（Nicardipine+Aβ1-40）和142.9±5.3%（AP-5+Aβ1-40）,明显低于Aβ1-40单独处理后的相对荧光强度值（P<0.01）。这表明,Aβ1-40引起的细胞内钙水平升高部分是通过L型电压门控钙通道（ L-type voltage-dependent calcium channels, L-VDCC）和NMDA （N-methyl-D-aspartate）受体介导的。（2）Val⁸-GLP-1（7-36）可剂量依赖性引起[Ca²⁺]i的一过性升高。给予培养细胞不同浓度的Val⁸-GLP-1（7-36） （10 nM、100 nM和1000 nM）后,[Ca²⁺]i相对荧光强度呈现剂量依赖性增强,给药后1分钟达到顶峰,然后逐渐下降并恢复到基础水平。我们测定了给药后1分钟10 nM、100 nM和1000 nM Val⁸-GLP-1（7-36）组的[Ca²⁺]i相对荧光强度值,分别为102.6±1.1%、126.5±4.5%和165.2±5.1%,与对照组相比,呈现逐渐升高趋势,尤其是100 nM和1000 nM Val⁸-GLP-1（7-36）引起的[Ca²⁺]i相对荧光强度值明显高于对照组（P<0.01）;然而,在给药后18分钟再次测定,以上各组的[Ca²⁺]i相对荧光强度值分别为99.6±1.6%、100.3±1.0%和100.1±0.54%,已经恢复到基础水平,与对照组没有差异（P>0.05）。（3）Val⁸-GLP-1（7-36）拮抗了Aβ1-40诱导的[Ca²⁺]i升高,并表现出一定程度的剂量依赖性。用不同浓度的Val⁸-GLP-1（7-36） （10 nM、100 nM和1000 nM）预处理培养的神经元约30 min后,随着Val⁸-GLP-1（7-36）浓度的升高,Aβ1-40诱导的[Ca²⁺]i升高效应逐渐减小。10 nM、100 nM和1000 nM Val⁸-GLP-1（7-36）预处理后,Aβ1-40（10μM）引起的相对荧光强度由单独给予Aβ1-40时的188.6±2.2%分别下降到165.6±3.4%、141.3±3.3%和123.6±3.5% （P<0.01）,表现出明显的剂量依赖性。以上结果表明:Aβ1-40可使大鼠原代培养的大脑皮层神经元[Ca²⁺]i升高,提示Aβ的神经毒性与Aβ引起的细胞内钙超载有关;Val⁸-GLP-1（7-36）剂量依赖性抑制了Aβ1-40诱导的[Ca²⁺]i升高,提示Val⁸-GLP-1（7-36）在行为学和电生理上表现出的神经保护作用,至少部分原因是通过减弱Aβ引起的细胞内钙超载实现的。总之,本研究利用Morris水迷宫、电生理场电位和脑片膜片钳、激光扫描共聚焦显微镜离子成像技术,通过观察大鼠空间学习记忆能力,记录在体海马L-LTP,引导脑片海马CA1区锥体神经元自发微突触后电流（mEPSCs和mIPSCs）以及测定原代培养大鼠皮层神经元细胞内钙离子水平的变化,探讨了Val⁸-GLP-1（7-36）对Aβ1-40神经毒性作用的调制以及可能机制。研究结果表明,Val⁸-GLP-1（7-36）能够有效对抗Aβ1-40诱导的大鼠空间学习记忆能力和在体海马L-LTP伤害,这种神经保护作用可能与Val⁸-GLP-1（7-36）对突触传递和细胞内钙稳态的精细调控有关。因而,本研究为GLP-1类似物Val⁸-GLP-1（7-36）的神经保护作用提供了进一步的行为学和电生理证据,并初步揭示了可能的细胞和离子机制,为开展GLP-1临床治疗AD奠定了一定的实验基础。更多还原

【Abstract】 Alzheimer disease （AD） is an age-related neurodegenerative disorder characterized by progressive learning impairment and memory loss followed with cognitive decline. The deposition of amyloidβ-protein （Aβ） in the brain, especially in the hippocampus and temporal cortex, is thought to be responsible for the deficit of learning and memory in AD patients. The neurotoxicity of Aβhas been widely reported in vivo and in vitro, including the impairment of spatial learning and memory and synaptic plasticity such as long-term potentiation （LTP）. Calcium ion is one of the most important intracellular second messengers in the brain, being essential for a variety of neuronal functions such as neuronal development, synaptic transmission and plasticity, and the regulation of various metabolic pathways. Accumulating evidence suggested that the disruption of intracellular Ca²⁺ homeostasis is crucial to Aβinduced neurodegeneration. However, it is seriously short of effective neuroprotective strategies against Aβneurotoxicity up to now.Interestingly, it has been found that type 2 diabetes mellitus （T2DM）, another degenerative disease, is a risk factor for developing AD in the elderly. One promising treatment for AD is using insulin-releasing gut hormone glucagon-like peptide-1 （GLP-1）, a modulator used in T2DM therapy. GLP-1 can reduce the levels of Aβin the brain in vivo and reduce levels of amyloid precursor protein （APP） in cultured neuronal cells and possess neurotrophic properties to protect neurons against glutamate-induced apoptosis. Previous study has shown that the hippocampal LTP detrimental effect induced by Aβfragments was effectively prevented by GLP-1. More importantly, GLP-1 and GLP-1 receptors （GLP-1R） are expressed in the brain, including the hippocampus. However, whether GLP-1 can protect against Aβ-induced impairment of synaptic plasticity, especially the late phase of long-term potentiation （L-LTP） in hippocampus, is still an open question; the mechanism of neuroprotective role of GLP-1 in the brain has not yet been fully elucidated at the present time. The natural GLP-1 can be rapidly degraded by the enzyme dipeptidyl peptidase IV （DPP IV） and its half life is only 2-3 min in blood plasma. In contrast, Val⁸-GLP-1（7-36） is a GLP-1 analogue with profound resistance to DPP IV and greater biological activity.Therefore, Val⁸-GLP-1（7-36）, not natural GLP-1, was used in the present study. By using the high-effective GLP-1 analogue, we investigated: （1） the effects of i.c.v. injection of Val⁸-GLP-1（7-36） on the Aβ1-40-induced impairment of spatial learning and memory of rats in a Morris water maze test; （2） the effects of i.c.v. injection of Val⁸-GLP-1（7-36） on the Aβ1-40-induced impairment of in vivo L-LTP in rat hippocampal CA1 region; （3） effects of Val⁸-GLP-1（7-36） on the Aβ1-40-induced changes in miniature postsynaptic currents （mEPSCs and mIPSCs） in hippocampal CA1 pyramidal neurons; （4） the effects of pretreatment with Val⁸-GLP-1（7-36） on Aβ1-40-induced elevation of [Ca²⁺]i in cultured primary rat cortical neurons.PartⅠVal⁸-GLP-1（7-36） Protects Against Aβ1-40-Induced Impairment of Spatial Learning and Memory in RatsTo characterize the neuroprotective role of Val⁸-GLP-1（7-36） in the brain, we investigated the effects of i.c.v. injection of Val⁸-GLP-1（7-36） on the Aβ1-40-induced impairment of spatial learning and memory of rats in a Morris water maze test and the alteration of CA1 neuronal morphology by HE staining after finishing the behavior experiment. The escape latency （s）, distance traveled （cm） and swimming speed （cm/s） were recorded in hidden platform tests, and the percentage of the total time in the different quadrants was calculated in probe trials.The results showed that: （1） i.c.v. injection of 5 nmol Aβ1-40 impaired the spatial learning and memory of rats. In hidden platform test, the latencies and distances for searching for the platform were significantly larger compared with control from the 2nd to the 5th day （P<0.01）. For example, the mean escape latencies and distances were 50±5.5 s and 1232.8±128.3 cm, significantly larger （P<0.01） than the values of 17.8±2.9 s as and 458.6±63.8 cm in the control group （P<0.01） at the 5th day of testing. In probe trial, the percentage of total time elapsed in the target quadrant in the Aβ1-40 group was only 28.4±3.8% in the Aβ1-40 group, significantly lower than 43.5±4.8% in control group （P<0.01）. （2） Val⁸-GLP-1（7-36） alone improved spatial learning and memory of rats in a dose-dependent manner. In hidden platform test, 0.05 pmol Val⁸-GLP-1（7-36） had no effects on the escape latencies and distances （p>0.05）; but 0.5 pmol and 5 pmol Val⁸-GLP-1（7-36） enhanced spatial learning ability of rats. In 5 consecutive days of testing, the latencies and distances were significant decreased in Val⁸-GLP-1（7-36） group compared with control （P<0.01）, for example, on the 5th day of testing, the escape latencies in 0.05 pmol, 0.5 pmol and 5 pmol Val⁸-GLP-1（7-36） groups were 15.5±1.4 s, 11.1±1.1 s and 6.8±1.1 s （p<0.01）, respectively, showing a dose-dependent decrease. In probe trial, the percentage of total time elapsed in the target quadrant in the 0.05 pmol, 0.5 pmol and 5 pmol Val⁸-GLP-1（7-36） groups were 44.9±4.4%, 48.5±4.9% and 51.4±3.8%, respectively, showing a significant increase with the increase of the concentration of Val⁸-GLP-1（7-36） （p<0.01）. （3） Pretreatment of Val⁸-GLP-1（7-36） effectively protected spatial learning and memory against Aβ1-40-induced impairment in a dose-dependent manner. For example, on the 5th day of hidden platform test, the escape latencies in 0.05 pmol, 0.5 pmol and 5 pmol Val⁸-GLP-1（7-36） plus Aβ1-40 groups were 30.3±3.5 s, 28.4±2.5 s and 18.8±2.6 s, respectively, showing a dose-dependent decrease. In probe trial, the percentages of total time elapsed in the target quadrant were 40.2±4.7%, 46.4±4.6% and 48.3±4.7% for 0.05 pmol, 0.5 pmol and 5 pmol Val⁸-GLP-1（7-36） puls Aβ1-40 group, respectively, significantly higher than Aβ1-40 alone group （p<0.01）, showing a significant dose-dependent increase （p<0.01）. （4） Both Val⁸-GLP-1（7-36） and Aβ1-40 did not affect the vision and the swimming speeds of approximately 19 cm/s. （5） The most pronounced lesions were found in the CA1 sector in Aβ1-40 group, including reduced pyramidal neurons, diminished neuron density, partial neuronal degeneration and necrosis. However, pretreatment with Val⁸-GLP-1（7-36） obviously reduced Aβ1-40-induced damage of neuron, and the pathological changes were gradually alleviated with the increase of the concentration of Val⁸-GLP-1（7-36）. These results indicated that i.c.v. administration of Aβ1-40 impaired spatial learning and memory of rats, while pretreatment with Val⁸-GLP-1（7-36） effectively reversed Aβ1-40-induced the impairment of cognitive function in a dose dependent manner, suggesting that natural GLP-1 in CNS may play an important positive role in maintaining normal cognitive function, and Val⁸-GLP-1（7-36） might be a promising strategy to ameliorate degenerative processes in AD.PartⅡVal⁸-GLP-1（7-36） Protects Against Aβ1-40-Induced Impairment of Hippocampal Late-Phase Long Term Potentiation in Rat Hippocampal CA1 Region in vivoThe effect of i.c.v. injection of Val⁸-GLP-1（7-36） on the Aβ1-40-induced impairment of in vivo L-LTP in rat hippocampal CA1 region was investigated in the present study. Hippocampal fEPSP were recorded in the CA1 region. Three sets of high frequency stimulation （HFS） with 5 min of interval were applied to produce a robust L-LTP.The results showed that: （1） Aβ1-40 did not affect the baseline fEPSP but significantly suppressed L-LTP. After application of 5 nmol Aβ1-40,the average fEPSP amplitudes before and 3 h after Aβ1-40 injection without HFSs were 99.5±1.4% and 100.2±1.11% （P>0.05）, respectively. However, the average fEPSP amplitudes after HFSs were 129.6±3.3%, 108.1±5.4% and 101.1±7.2% in Aβ1-40 group at 1 h, 2 h and 3 h, respectively, significantly lower than the values of 153.0±4.5%, 140±4.8% and 139.38±5.5% in control group at the same time points （P<0.01）. （2） Val⁸-GLP-1（7-36） alone did not affect the baseline fEPSP but enhanced the L-LTP in a dose-dependent manner. The baseline average fEPSP amplitudes induced by test stimulation before and 3 h after injection were 99.5±1.4% and 100.2±1.1% （P>0.05）, 100.2±2.4% and 99.5±4.7% （P>0.05）, 99.9±3.32% and 100.0±8.6% （P>0.05） in 0.05 pmol, 0.5 pmol and 5 pmol Val⁸-GLP-1（7-36） group （P>0.05）, respectively. However, the averaged fEPSP amplitudes after HFSs were gradually increased with the increase of the concentration of Val⁸-GLP-1（7-36）. For example, the averaged fEPSP amplitudes were 143.2±7.1%, 152.2±2.4% and 164.0±4.1% in the 0.05 pmol, 0.5 pmol and 5 pmol Val⁸-GLP-1（7-36） group at 3 h post-HFSs, respectively, significantly larger than the values in control group （143.2±1.5%, P<0.01） （3） Pretreatment with Val⁸-GLP-1（7-36） protected against Aβ1-40-induced impairment of L-LTP. The average fEPSP amplitudes in 0.05 pmol, 0.5 pmol and 5 pmol Val⁸-GLP-1（7-36） plus Aβ1-40 group were 130.3±6.0%, 138.9±6.0% and 139.0±5.3% at 3 h post-HFSs, respectively, significantly larger than the values in Aβ1-40 alone group （P<0.01）.These results demonstrated that pretreatment with Val⁸-GLP-1（7-36） effectively reversed Aβ1-40-induced suppression of L-LTP in a dose-dependent manner in the rat hippocampal CA1 region in vivo. The results are well consistent with the results in the behavior experiment above and may partly explain the cellular mechanisms of GLP-1 in improving spatial learning and memory function.PartⅢEffects of Val⁸-GLP-1（7-36） on the Spontaneous Synaptic Activity in Hippocampal SlicesThe proper modulation of excitatory and inhibitory synaptic transmission is critical for brain function. To further reveal the cellular electrophysiological mechanism of neuroprotective role of Val⁸-GLP-1（7-36）, we, in this part of experiment, examined spontaneous miniature postsynaptic currents （mEPSCs and mIPSCs） in hippocampal CA1 pyramidal neurons of rat brain slices by using whole-cell patch clamp technique.The results showed that: （1） Aβ1-40 significantly reduced the frequency of mEPSCs and mIPSCs and extended the decay time of mIPSCs. After application of 100 nM Aβ1-40 alone, the normalized frequency of mEPSCs reduced from 100.0±11.2% in control group to 37.9±8.8% （P<0.01）; the normalized frequency of mIPSCs reduced from 100.0±5.1% in control group to 46.5±6.8% （P<0.01）. However, the amplitude, 10-90% rise time and decay time of mEPSCs and the amplitude, 10-90% rise time of mIPSCs in Aβ1-40 group did not change （P>0.05）. （2） Val⁸-GLP-1（7-36） （10 nM） did not change the amplitude, frequency, 10-90% rise time and decay time of mEPSCs and mIPSCs （P>0.05）. （3） Pretreatment with Val⁸-GLP-1（7-36） improved Aβ1-40-induced change in frequency of mEPSCs and mIPSCs and decay time of mIPSCs. After pretreatment with Val⁸-GLP-1（7-36）, the normalized frequency of mEPSCs significantly increased to 63.5±5.0% from 37.9±8.8% in Aβ1-40 alone group （P<0.01）; the frequency of mIPSCs increased to 72.4±2.2% from 46.5±6.8% in Aβ1-40 alone group （P<0.01）; the normalized decay time of mIPSCs decreased to 124.1±10.8% from 150.2±13.4% in Aβ1-40 group alone （P<0.01）.These results clearly demonstrated that pretreatment of Val⁸-GLP-1（7-36） contributed to Aβ1-40 induce decrease of frequency of mEPSCs and mIPSCs and change of mIPSCs channel kinetics. These results may be helpful in explaining the mechanism of Aβ1-40 induced impairment in hippocampal synaptic plasticity. The decrease of the frequency of spontaneous mEPSCs will affect the presynaptic transmitter release, and the decrease of the frequency of spontaneous mIPSCs may cause the balance between excitation and inhibition of nervous system and enhance the cell excitotoxicity. Therefore, the effects of Val⁸-GLP-1（7-36） on the Aβ1-40-induced changes of mEPSCs and mIPSCs are positive in the regulation of neuronal activity, and GLP-1 and its analogue may represent an alternative and potentially valuable novel therapeutic intervention for reversing the neurodegenerative processes in AD.PartⅣEffects of Val⁸-GLP-1（7-36） on Aβ1-40-Induced Calcium Influx in Cultured Primary Rat Cortical NeuronsBy utilizing calcium imaging via laser-scanning confocal fluorescent imaging technique, we investigated the effects of Aβ1-40 and Val⁸-GLP-1（7-36） on [Ca²⁺]i, especially the neuroprotective effects of Val⁸-GLP-1（7-36） against Aβ1-40-induced disruption of Ca²⁺ homeostasis in cultured primary rat cortical neurons.The results showed that: （1） Aβ1-40 alone significantly increased the intracellular calcium level. 18 min after application of 10μM Aβ1-40, the relative fluorescent intensity of cultured primary rat cortical neurons obviously increased from 100% in control to 191.1±21.6% （P<0.01）. Pretreatment with 50μM AP-5, a specific inhibitor of NMDA receptor, or 5μM Nicardipine, a specific inhibitor of L-VDCC, suppressed Aβ1-40-induced [Ca²⁺]i elevations, with a relative fluorescent intensity of 142.9±5.3% and 152.1±1.7% （P<0.01）, respectively. These results showed that Aβ1-40-induced [Ca²⁺]i elevations are mediated partly by L-type voltage-dependent calcium channels （L-VDCC） and N-methyl-D-aspartate （NMDA） receptor. （2） Val⁸-GLP-1（7-36） alone induced a transient elevation of [Ca²⁺]i in a dose-dependent manner. The fluorescent intensity of [Ca²⁺]i after application of different concentration of Val⁸-GLP-1（7-36） rapidly rised to peak values at 1 min after administration in a dose-dependent manner. The relative fluorescent intensity of [Ca²⁺]i were 102.6±1.1%, 126.5±4.5% （P<0.01） and 165.2±5.1% （P<0.01） in 10 nM, 100 nM and 1000 nM Val⁸-GLP-1（7-36） group, respectively, showing a dose-dependent increase （p<0.01）. However, at the time point of 18 min after application, the relative fluorescent intensity of [Ca²⁺]i returned to baseline, being 100.1±0.54%, 100.3±1.0% and 99.6±1.6% in 10 nM, 100 nM and 1000 nM Val⁸-GLP-1（7-36） group, respectively. （3） Pretreatment with Val⁸-GLP-1（7-36） protected against Aβ1-40-induced elevation of [Ca²⁺]i in a dose-dependent manner. The relative fluorescent intensity of [Ca²⁺]i decreased to 165.6±3.4%, 141.3±3.3% and 123.6±3.5% in co-application of 10 nM, 100 nM and 1000 nM Val⁸-GLP-1（7-36） puls Aβ1-40 group, respectively, significantly lower than the value of 188.6±2.2% in Aβ1-40 alone group （P<0.01）.These results demonstrated that Aβ1-40 alone significantly increase the intracellular calcium levels, which is closely related to the neurotoxicity of Aβ-induced calcium overload. The fact that pretreatment with Val⁸-GLP-1（7-36） effectively protected against Aβ1-40-induced elevation of [Ca²⁺]i in a dose-dependent manner suggested that the neuroprotective effects of Val⁸-GLP-1（7-36） in the behavioral and electrophysiological studies may result from the alleviation of Aβ-induced intracellular calcium overload.In conclusion, the present study, by using Morris water maze test, field potential recording, brain slice whole-cell patch clamp and confocal Ca²⁺ image technique, observed the effects of Val⁸-GLP-1（7-36） on the spatial learning and memory behavior, hippocampal L-LTP in vivo, spontaneous miniature postsynaptic currents （mEPSCs and mIPSCs） and [Ca²⁺]i of cultured cortical neurons of rats, and investigated the neuroprotective role of Val⁸-GLP-1（7-36） against Aβ1-40-induced neurotoxicity. The results indicated that Val⁸-GLP-1（7-36） can effectively protect against Aβ1-40-induced impairment of rat spatial learning and memory and hippocampal L-LTP in vivo; the neuroprotective effect of Val⁸-GLP-1（7-36） may be related to the modulation of synaptic transmission and intracellular calcium homeostasis. Therefore, the present study provides further behavioral and electrophysiological evidence for the neuroprotective effect of GLP-1 and its analogues Val⁸-GLP-1 （7-36）, and reveals its possible cellular and channel mechanism, strongly suggesting that GLP-1 and its analogue might be one of the promising candidates for the treatment of AD in the future.更多还原