Name: modeling amyloid-β42 toxicity and neurodegeneration in adult zebrafish brain
Uploaded: 2017-10-25T12:30:00.000+00:00
Duration: 10 min 1 s
Description: Watch this Scientific Journal Video about Modeling Amyloid-Î²42 Toxicity and Neurodegeneration in Adult Zebrafish Brain at JoVE.com

这项工作得到了 DZNE 和亥姆霍兹协会 (VH-NG-1021)、》、TU 德累斯顿 (FZ-111、043_261518) 和 DFG (KI1524/6) (酸) 的支持;由莱布尼茨协会 (SAW-2011-IPF-2) 和 BMBF (BioLithoMorphie 03Z2E512) (Y.Z.)。我们还要感谢乌莉霍夫曼肽合成, 南 Asokan, Prayag Murawala, 和艾利在拍摄过程中帮助田中。

本议定书是欧盟准则 (2010/63) 和欧洲生物和医学鱼类模型协会 (EuFishBioMed) 在卡尔斯鲁厄中国科学院技术 (KIT) 中建议的标准程序。所有在这里描述的方法都得到了道德委员会的批准 (Landesdirektion 德累斯顿; 文档编号 TVV-52/2015). 
 1. 制备 #946; 42 肽 <ol> <li> 合成多肽 (参见 表 1 ) 使用标准的 9-芴 (甲氧羰基) 化学与 2-(1 h-benzotriazol-1-基)-11, 33-tetramethyluronoiumhexafluorphosphate (HBTU) 作为自动固相多肽合成器的偶联试剂 52 。合成的规模是100和 #181; 摩尔. </li>
	<li> 将 n-芴甲氧羰基保护树脂的500毫克的自动多肽合成器加载为固相 (负载容量为0.2 摩尔/克)。装载被溶化的 n-芴甲氧羰基被保护的氨基酸在集中 0.5 M 在需要的容量和为各自合成器计算. 
	注: 进行了计算, 使每块 n-芴甲氧羰基被保护的氨基酸的偶联度为每一个积木的5倍, 超过了树脂的 47 . </li>
	<li> 溶解 dimethlyformamide (DMF) 中所需的多肽合成试剂。例如, 准备活化剂, HBTU, 在浓度为0.48 米, 45% 伏/v n-啉 (NMM) (基地), 和5% 伏/v 醋酸酐 (上限混合物) 上限的 non-reacted 氨基. </li>
	<li> 将所有的肽从树脂中通过连续混合的固体支持使用搅拌器在一个新准备的裂解混合物组成的乙酸酸 (TFA): Triisopropylsilane (TIS): 水: 糖 (数码) 在 90 (v/v): 5 (v/v): 2。5(五/五): 2.5 (m/五), 4 h. 为100和 #181 的合成规模使用10毫升; 摩尔. </li>
	<li> 通过将裂解混合物加入100毫升 ice-cold 二乙醚, 沉淀所裂解产物。通过含有聚四氟乙烯 (PTFE) 过滤器的过滤单元, 其孔径大小为0.45 和 #181; m 和20毫升 ice-cold 乙醚洗涤. </li>
	<li> 从滤纸中收集过滤后的肽, 在5毫升蒸馏水中溶解100毫克沉淀肽: 乙腈在 1:1. </li>
	<li> 通过反相高压液相色谱法 (hplc) 对 semi-preparative 的多孔聚苯乙烯苯柱尺寸10和 #181; m. 
	<ol> <li> 预热该列并保持在50和 #176; C 使用柱式加热装置。收集所有主要分数使用自动分数收集器, 应用梯度从5% 到100% 溶剂 B 超过25分钟, 在4毫升/分钟. 
		注: 溶剂 a 是 0.1% TFA 在水和溶剂 B 是 0.1% TFA 在乙腈 52 . </li>
		<li> 监测色谱在 220 nm, 收集适当的峰值, 并使用 LC-MS 进行分析. </li>
	</ol> </li> <li> 通过分析 C18 柱 (珠粒大小1.7 和 #181; m) 通过检测, 通过对紫外线探测器的分析, 在 220 nm 上通过解析反相超高压液相色谱 (UPLC) 来确认纯度。用质谱法确认多肽产品. 
	注: UPLC 是耦合到一个电喷雾电离质谱 (ESI-MS) 和串联四极探测器. </li>
	<li> 通过将0.052 毫巴的真空度应用到蓬松的粉末上, 将所需肽的正确分数 Lyophilize 在圆底烧瓶中。将真空泵耦合到-78 和 #176 的冷冻装置上; c.-80 和 #176; c, 无限期存储. </li>
	<li> 使用冻干肽在乙腈混合物中制备1毫米的库存溶液: DMF: 分析级水在 1:1: 1 用于实验。此解决方案可以存储至少6月, 因为该解决方案不聚合多肽. </li>
</ol> 2。喷射混合物的制备 <ol> <li> 准备磷酸盐缓冲盐水 (PBS) (137 毫米氯化钠, 2.7 毫米氯化钾, 10 mm Na 2 HPO 4 , 2 mm, 2 PO 4 , pH 7.4) 用于稀释冻干肽. </li>
	<li> 将该肽分解为20和 #181 的最终浓度; M 在 PBS 中。准备这个解决方案新鲜。混合好, 并在冰上储存, 直到注射。不要超过30分钟, 以防止在解决方案中聚合. </li>
</ol> 3。麻醉 <ol> <li> 准备麻醉剂的储存液, 0.1% 乙基氨基 methanesulphonate (MESAB), 从循环系统中的常规鱼水中。准备 anaesthetization 溶液, 最终浓度为 0.0025% (v/v). </li>
	<li> 将所需数量的鱼从其罐中移到一个带有5升系统水的运输容器中. </li>
	<li> 半填充一个塑料培养皿 (90 毫米) 与40毫升麻醉剂。用这个盘子做注射. </li>
	<li> 将鱼放入麻醉剂中, 直到盖运动停止. </li>
</ol> 4。脑室微注射 <ol> <li> 注射装置的制备 <ol> <li> 使用针牵引器制备玻璃注射毛细管, 其参数如下: 加热循环, 537; 牵引循环, 250; 速度1.5 s;时间, 80 毫秒 </li> <li> 将压力源上的压力设置为 25 psi. </li>
		<li> 将微量参数设置为: 按住压力 20 psi; 弹出压力 10 psi; 周期值 2.5; 浇注值100毫秒 </li>
		<li> 将玻璃毛细管与注射液一起加载。将玻璃毛细管插入微注射支架。将注塑角度调整为45和 #176;. 
		注意: 可以找到更详细的协议, 如参考资料 27 , 46 中所述。
	</ol> </li> <li> 将一条鱼放入一个充满 anaesthetization 解决方案的新培养皿中 (如步骤3.3 中所示). </li>
	<li> 拿着钳子的鱼和东方注射. </li>
	<li> 用30克针的尖端在光学顶上产生一个狭缝, 两个侧面板相遇。不要将笔尖插入脑组织, 这会导致出血和损伤。有关其他详细信息, 请参阅参考 27 、 45 、 46 。
	<li> 将玻璃毛细管插入狭缝中. 
	<ol> <li> 仅使用针头的尖端, 并且不穿透头骨超过1毫米。保持鱼和插入玻璃毛细管的尖端通过切口部位. </li>
		<li> 将玻璃毛细管的尖端朝向端的45和 #176; 角度。注入1和 #181; 我的解决方案。液体在注射后立即散开. </li>
	</ol> </li> </ol> 5。恢复 <ol> <li> 将鱼放回传输容器中, 直到它恢复为止。将容器与定期循环的鱼水连接, 以确保最佳的水质. 
	注意: 恢复通常需要1分钟。如果需要更长的时间, 必须将鱼放在麻醉剂中较短 (这需要由实验者进行优化). </li>
</ol > 6。组织准备和切片 <ol> <li> 在牺牲鱼之前等待一段希望的时间. 
	注意: 这取决于实验问题。淀粉样蛋白的沉积可以早在注射后1天就能看到. </li>
	<li> 用适当的方法牺牲鱼根据道德规则 ( 例如 , 对待鱼与 0.1 M MESAB). </li>
	<li> 将顶上的颅骨切开, 用尖头 cep 在背侧, 用手术刀解剖胸肋后方的头部. </li>
	<li> 在4和 #176 一夜之间使用2% 甲醛 (粉煤灰) 固定磁头; c. 使用3毫升的粉煤灰每头在一个塑料管与螺丝盖. 
	注意: 在处理粉煤灰时, 应佩戴适当的个人防护设备. </li>
	<li> 对于冻和脱, 洗头三次在0.1M 磷酸盐缓冲 (pH 值 7.4), 然后转移到 20% sucrose/20%ethylenediaminetetraacetic (EDTA) 溶液和孵化过夜在4和 #176; C. </li>
	<li> 将组织嵌入到切片树脂中, 将 7.5% gelatin/20%sucrose 溶液中的头冻结在干冰 (大约3-5 分钟以冻结块) 的塑料组织学模子中。将示例存储在-80 和 #176; C 或继续下一步 (cryosectioning). </li>
	<li> 将磁头分为12和 #181; m 厚 cryosections 使用低温描述的 28 , 42 。将 cryosections 直接转移到玻片上, 并存放在-20 和 #176; C 用于长期使用. </li>
</ol> 7。免疫组织化学染色与显微术 注意: 在湿化室中执行所有孵化步骤。所有的洗涤步骤为10分钟. 
<ol> <li> 在室温下将部分干燥30分钟。解冻后, 清洗两次在 pbs 和一次在 PBSTx (pbs 与 0.03% Triton-X-100). </li>
	<li> 用主抗体 (抗 A 和 #946; 42 抗体; 1:500 稀释在 PBSTx) 在4和 #176; C 隔夜. 和 #160; 第二天, 在 PBS 洗一次, 两次在 PBSTx. </li>
	<li> 在室温下, 用二次抗体 (荧光耦合检测, 1:500 稀释) 和 DAPI (1 和 #956; g/毫升) 在 PBSTx 中孵育剖面, 2 小时. </li>
	<li> 在 PBSTx 中洗三次。在最后的洗涤之后, 用片和 #160 安装幻灯片; 使用100和 #956; L 70% 甘油. </li>
	<li> 使用共聚焦显微镜获取荧光图像 (例如, 20X PL 0.7 目标具有488励磁范围和标准绿色滤镜; 图 2 ) 39 . </li> </ol>

作者没有透露

淀粉样肽可以被修改, 包括序列变化或各种标签。例如, 可以生成一个被炒的淀粉样肽, 多肽可以在肽末端的 N 端标上荧光标记, 或用载体肽39标记。同样, 在这个协议中, 载体肽是细胞穿透肽 TR, 因为它的有效运输货物深入到脑组织39。此外, 我们的方法允许注射和分析各种多肽, 可能导致有毒的聚合50,51。因此, 我们的系...

AD 是一种慢性渐进性疾病, 其特点是大脑皮层神经元和突触的丢失1,2,3,4,5。经典的病理标志的广告是淀粉样肽的沉积和形成的纤维缠结 (NFTs)6。老年斑块, 也称为淀粉样斑块, 是由淀粉样β (a) 肽组成的β褶结构在脑实质5。Aβ42在 AD 患者中的积累在疾病进展中具有早期和关键性的作用。AD 触发一连串的事件导致突触功能障碍, 可塑性受损, 神经元丢失7,8,9,10。
硬斑马鱼的成年大脑是研究干细胞可塑性调控的优良模型 11,12,13,141516、17、18、19、20以及中枢神经系统中的各种疾病 (包括 AD21、22、23 ,24。由于大量可用的实验方法 19,20,25,26,27,28,2930,31, 这些研究是翔实和可行的。斑马鱼可以补充中枢神经系统13,15,32,33,34,35,36,37, 38, 部分使用在神经元丢失后激活的分子程序19,39,40,4142,43, 44。因此, 在斑马鱼中建立一个神经退行性疾病模型可以帮助解决脊椎动物大脑再生能力和干细胞生物学方面的新问题。
最近, 我们通过注射合成 Aβ42肽 (表 1)39在成年斑马鱼脑中建立了淀粉样毒性模型。这种注射引起了神经表型的人脑病理学 (例如, 细胞死亡, 胶质活化, 突触变性, 和记忆缺陷), 表明斑马鱼可用于诱导神经在斑马鱼脑中, Aβ42肽可通过免疫组化染色检测, 并可识别成年斑马鱼中枢神经系统再生的分子机制39。在本协议中, 我们演示了使用脑室注射液 (CVMI) 方法27,39,45,46 , 将合成的淀粉样肽注射到斑马鱼脑中。模拟淀粉样蛋白沉积 (图 1)。CVMI 提供了一种新的方式提供的多肽, 其聚合后注射作为β板结构和施加毒性。这些肽分布在整个大脑中, 以整个 rostro 尾轴的心室面积为目标45。此外, 该方法还可以分析 NSPCs 在成年斑马鱼脑内淀粉样蛋白包裹体中的形态和分子反应。这些研究将为我们提供一个成功的哺乳动物大脑修复的洞察力。我们的方法可以用来理解的必要分子机制, 成功的再生反应后, AD 样症状, 以诱导补充的丢失神经元和功能恢复。

<table><tbody><tr><td>Fmoc-protected amino acids</td><td>IRIS Biotech GmbH (Marktredwitz, Germany)</td><td></td><td>Fmoc-based amino acids for solid phase peptide synthesis (SPPS)</td></tr><tr><td>N,N,N&amp;prime;,N&amp;prime;-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU)</td><td>IRIS Biotech GmbH (Marktredwitz, Germany)</td><td>RL-1030</td><td>Activator</td></tr><tr><td>Oxyma</td><td>IRIS Biotech GmbH (Marktredwitz, Germany)</td><td>RL-1180</td><td>Racemization supressor</td></tr><tr><td>N,N-Diisopropylethylamine</td><td>IRIS Biotech GmbH (Marktredwitz, Germany)</td><td>SOL-003</td><td>Base</td></tr><tr><td>Dimethylformamide</td><td>IRIS Biotech GmbH (Marktredwitz, Germany)</td><td>SOL-004</td><td>Solvent</td></tr><tr><td>N-Methylmorpholine</td><td>Thermo Fisher (Kandel) GmbH, Germany</td><td>A12158</td><td>Base</td></tr><tr><td>1-Hydroxybenzotriazole hydrate (HOBT)</td><td>Sigma-Aldrich Co. LLC. (St. Louis, MO, USA)</td><td>157260 ALDRICH</td><td>Activator</td></tr><tr><td>Piperidine</td><td>MERCK KGaA (Darmstadt, Germany)</td><td>822299</td><td>Fmoc deprotection reagent</td></tr><tr><td>Dichlormethane (DCM)</td><td>MERCK KGaA (Darmstadt, Germany)</td><td>106050</td><td>Solvent</td></tr><tr><td>Formic acid (FA)</td><td>MERCK KGaA (Darmstadt, Germany)</td><td>100264</td><td>Buffer component for HPLC</td></tr><tr><td>Trifluoroacetic acid (TFA)</td><td>MERCK KGaA (Darmstadt, Germany)</td><td>808260</td><td>Clevage Mixture reagent</td></tr><tr><td>Triisopropylsilane(TIS)</td><td>MERCK KGaA (Darmstadt, Germany)</td><td>233781 ALDRICH</td><td>Clevage Mixture reagent</td></tr><tr><td>Acetonitrile (for UPLC/LCMS)</td><td>Sigma-Aldrich Laborchemikalien GmbH</td><td>34967-1L</td><td>Solvent</td></tr><tr><td>Acetonitrile (for HPLC)</td><td>VWR International Ltd, England</td><td>83639.320</td><td>Solvent</td></tr><tr><td>Diethylether</td><td>VWR International Ltd, England</td><td>23811.326</td><td>Solvent for peptide precipitation</td></tr><tr><td>Dithiotritol (DTT)</td><td>VWR International Ltd, England</td><td>0281-25G</td><td>Clevage Mixture reagent</td></tr><tr><td>TentaGel S RAM Fmoc rink amide resin</td><td>Rapp Polymere GmbH (Tuebingen, Germany)</td><td>S30023</td><td>Solid phase for SPPS</td></tr><tr><td>Peptide synthesis 5 ml syringes with included filters</td><td>Intavis AG (Cologne, Germany)</td><td>34.274</td><td>Reaction tube for SPPS and for clevage from the Solid Phase</td></tr><tr><td>Polytetrafluoroethylene (PTFE) filter</td><td>Sartorius Stedtim (Aubagne, France)</td><td>11806-50-N</td><td>Filteration of precipitated peptides</td></tr><tr><td>Polyvinylidenefluoride (PVDF) syringe filter</td><td>Carl Roth GmbH + Co. KG Karlsruhe</td><td>KC78.1</td><td>Pre-filteration for HPLC</td></tr><tr><td>Peptide Synthesizer</td><td>Intavis, Cologne, Germany</td><td>ResPep SL</td><td>Automated solid-phase peptide synthesizer</td></tr><tr><td>Water Alliance HPLC</td><td>Waters, Milford Massachusetts, USA</td><td>Waters 2998, Waters e2695</td><td>Semi-preparative reverse-phase high pressure liquid chromatography (HPLC)</td></tr><tr><td>PolymerX, bead size 10&amp;mu;m, 250x10 mm</td><td>Phenomenex Ltd. Germany</td><td>00G-4328-N0</td><td>Porous polystyrene divinylbenzene HPLC column</td></tr><tr><td>Milli-Q Advantage A10, with a Milli-Q filter</td><td>EMD Millipore Corporation, Billerica, MA, USA</td><td>LCPAK0001</td><td>Water purification system</td></tr><tr><td>Filtration Unit</td><td>Sartorius Stedtim (Aubagne, France)</td><td>16307</td><td>Filtration unit for peptide precipitation</td></tr><tr><td>UPLC Aquity with UV Detector</td><td>Waters, Milford Massachusetts, USA</td><td>M09UPA 664M</td><td>Analytical reverse phase ultra HPLC for LC-MS</td></tr><tr><td>ACQUITY UPLC BEH C18, bead size 1.7 &amp;mu;m, 50x2.1 mm</td><td>Waters, Milford Massachusetts, USA</td><td>186002350</td><td>Analytical C18 column</td></tr><tr><td>ACQUITY TQ Detector</td><td>Waters, Milford Massachusetts, USA</td><td>QBB908</td><td>Electrospray ionization mass spectrometry (ESI-MS)</td></tr><tr><td>CHRIST ALPHA 2-4 LD plus + vacuubrand RZ6</td><td>Martin Christ Gefriertrocknungsanlagen GmbH, Germany</td><td>16706, 101542</td><td>Lyophilizer with vaccum pump</td></tr><tr><td>Paradigm plate reader</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>MESAB (ethyl-m-aminobenzoate methanesulphonate)</td><td>Sigma-Aldrich</td><td>A5040</td><td></td></tr><tr><td>Petri dishes</td><td>Sarstedt</td><td>821.472</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td>Life Technologies, GIBCO</td><td>10010-056</td><td></td></tr><tr><td>Needle</td><td>Becton-Dickinson</td><td>305178</td><td></td></tr><tr><td>Dissecting microscope</td><td>Olympus, Leica, Zeiss</td><td>Varies with the manufacturer</td><td></td></tr><tr><td>Dumont Tweezers</td><td>World Precision Instruments</td><td>501985</td><td></td></tr><tr><td>Gillies Dissecting Forceps</td><td>World Precision Instruments</td><td>501265</td><td></td></tr><tr><td>Glass injection capillaries</td><td>World Precision Instruments</td><td>TWF10</td><td></td></tr><tr><td>PicoNozzle</td><td>World Precision Instruments</td><td>5430-12</td><td></td></tr><tr><td>Pneumatic PicoPump</td><td>World Precision Instruments</td><td>SYS-PV820</td><td></td></tr><tr><td>Ring illuminator; Ring Light Guide</td><td>Parkland Scientific</td><td>ILL-RLG</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM1950</td><td></td></tr></tbody></table>

modeling amyloid-&#946;42 toxicity and neurodegeneration in adult zebrafish brain

阿尔茨海默氏病 (AD) 是一种衰弱的神经退行性疾病, 其中有毒 amyloid-β42 (Aβ42) 肽的积累导致突触变性、炎症、神经元死亡和学习缺陷。由于神经干/祖细胞 (NSPCs) 的增殖能力受损, 神经元发生减少, 在 AD 的情况下, 人类无法再生丢失的神经细胞。因此, 有效的再生疗法也应提高 NSPCs 的增殖和神经源能力. 斑马鱼 (斑马鱼) 是一种再生有机体, 我们可以学习基本的分子程序, 我们可以设计处理 AD 的治疗方法。因此, 在斑马鱼中生成类似广告的模型是必要的。利用我们的方法, 我们可以引入 Aβ42肽的合成衍生物, 将组织穿透能力纳入成年斑马鱼脑, 并分析疾病病理和再生反应。比现有的方法或动物模型的优势是, 斑马鱼可以教我们如何一个脊椎动物的大脑可以自然再生, 从而帮助我们更好地治疗人类神经退行性疾病的目标内生 NSPCs。因此, 在成年斑马鱼脑中建立的淀粉样毒性模型可能为神经科学和临床医学领域的研究开辟新的途径。此外, 这种方法的简单执行, 允许 cost-effective 和有效的实验评估。这份手稿描述了 Aβ42肽的合成和注射到斑马鱼脑。

采用高效液相色谱法纯化了合成的多肽, 并利用质谱法对纯化的淀粉样β肽进行了表征。将 HPLC 柱加热至50° c, 以改善 a 肽的分离, 并收集所有的分数。为了识别正确的合成肽, 对所有的分数进行了质谱分析。UPLC 色谱显示了化合物的纯度。在 UPLC 上产生一个峰值的 HPLC 分数 (即, 所需的淀粉样β肽的正确质量比) 进一步处理实验 (图 2)。
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Watch this Scientific Journal Video about Modeling Amyloid-Î²42 Toxicity and Neurodegeneration in Adult Zebrafish Brain at JoVE.com

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

本协议描述了单体 amyloid-β42肽在成年斑马鱼中产生淀粉样毒性的合成、表征和注射, 以建立阿尔茨海默病模型, 随后进行组织学分析和检测聚合.

成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

modeling-amyloid-42-toxicity-neurodegeneration-adult-zebrafish

Research

JoVE Journal

Neuroscience

11.1K 次观看.  German Centre for Neurodegenerative Diseases (DZNE) Dresden within Helmholtz Association. 本协议描述了单体 amyloid-β42肽在成年斑马鱼中产生淀粉样毒性的合成、表征和注射, 以建立阿尔茨海默病模型, 随后进行组织学分析和检测聚合.

视频: Modeling Amyloid-β42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological problems after stroke and is shown to impair neurogenic processes. Interestingly, the adult zebrafish has recently emerged as a relevant and useful model to mimic hyperglycemia/diabetes and to investigate constitutive and regenerative neurogenesis. This work provides methods to develop zebrafish models of hyperglycemia to explore the impact of hyperglycemia on brain cell proliferation under homeostatic and brain repair conditions. Acute hyperglycemia is established using the intraperitoneal injection of D-glucose (2.5 g/kg bodyweight) into adult zebrafish. Chronic hyperglycemia is induced by immersing adult zebrafish in D-glucose (111 mM) containing water for 14 days. Blood-glucose-level measurements are described for these different approaches. Methods to investigate the impact of hyperglycemia on constitutive and regenerative neurogenesis, by describing the mechanical injury of the telencephalon, dissecting the brain, paraffin embedding and sectioning with a microtome, and performing immunohistochemistry procedures, are demonstrated. Finally, the method of using zebrafish as a relevant model for studying the biodistribution of radiolabeled molecules (here,[18F]-FDG) using PET/CT is also described.

This work describes methods to establish acute and chronic hyperglycemia models in zebrafish. The aim is to investigate the impact of hyperglycemia on physiological processes, such as constitutive and injury-induced neurogenesis. The work also highlights the use of zebrafish to follow radiolabeled molecules (here, [18F]-FDG) using PET/CT.

斑马鱼高血糖的急性和慢性模型:评估高血糖对神经发生的影响和放射性标记的分子的生物分布的方法

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological ...

科研

发育生物学

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

环境污染物对斑马鱼幼虫的神经行为影响研究

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

环境科学

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or cognitive impairment. We have modified and developed a three-chamber choice cognitive task similar to that used with rodents for use with hyperglycemic zebrafish. The testing chamber consists of a centrally located starting chamber and two choice compartments on either side, with a shoal of conspecifics used as the reward. We provide data showing that once acquired, zebrafish remember the task at least 8 weeks later. Our data indicate that zebrafish respond robustly to this reward, and we have identified cognitive deficits in hyperglycemic fish after 4 weeks of treatment. This behavioral assay may also be applicable to other studies related to cognition and memory.

We present a behavioral chamber designed to assess cognitive performance. We provide data showing that once acquired, zebrafish remember the task 8 weeks later. We also show that hyperglycemic zebrafish have altered cognitive performance, indicating that this paradigm is applicable to studies assessing cognition and memory.

以斑马鱼为模型系统的三室选择行为任务

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or ...

行为学

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

触发细胞应激和死亡使用传统的紫外线激光共聚焦显微镜

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

神经科学

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

在体内病态的人类基因组建模使用斑马鱼

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

生物学

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.

研究钝力损伤对成年斑马鱼影响的可扩展模型

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

穿梭盒测定作为使用成人斑马鱼进行学习和记忆研究认知评估的关联学习工具

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

脑室微注射脂多糖到斑马鱼幼虫中以评估神经炎症和神经毒性

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...

行为的：评估成年斑马鱼神经毒性作用的测试电池

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

作者聚焦：成年斑马鱼环境和药物诱导的行为变化评估 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.