这项工作是由神经疾病研究所的支持和中风授予NS081333（至CMT），阿尔茨海默氏症协会授予NIRG-10-171721和心理健康补助MH096702研究所（UH到），以及国家老龄问题研究所资助的阿尔茨海默氏症研究中心在哥伦比亚大学授予试点AG008702（以YYJ和JB）。

注:对于所有的动物实验，体制和照顾和使用实验动物的国家准则随访。 1.准备手术器械和解决方案手术<ol><li>高压灭菌全不锈钢手术器械。 </li><li>通过稀释200标准的无水乙醇，用无菌分子级去离子蒸馏水制备70％的乙醇。 </li><li>装上29克针的汉密尔顿注射器。通过反复制定和喷射超纯水1分钟清洁Hamilton注射器和针头的内部。重复此过程，用70％的乙醇。 <ol><li>取下汉密尔顿注射器的活塞和针头。空气干燥在层流罩O / N的部分。 </li><li>照射的针头和注射器用紫外线灯使用前30分钟。 </li></ol></li><li>通过在分子级水中溶解的NaCl至最终重量的0.9％的体积浓度制备盐水溶液。 Sterili通过0.2微米的微孔过滤器过滤它泽解决方案。 </li></ol> 2.准备低聚Aβ1-42 <ol><li> Monomerize，重悬重组人的Aβ1-42在DMSO中的5mM完全按法"和其他13的出版物中描述。 </li><li>稀释5毫米Aβ1-42用无菌（1X）PBS以100μM在手术前一天。 </li><li>通过研磨混合解决方案很好。 </li><li>孵育的Aβ1-42溶液12小时，在4℃。 注意:控制解决方案，如在PBS稀释DMSO或加扰/反向的Aβ1-42肽应准备方式相同的Aβ1-42。 </li></ol> 3.确定注射坐标<ol><li>使用成年小鼠脑图谱来确定确切的前-后（AP），内侧-外侧（ML），和背腹（DV）的坐标为感兴趣14的大脑区域。 没有TE:为了说明我们选择了海马齿状回为目标与前囟门以下坐标技术:AP，-2.00毫米; ML，±1.3毫米; DV，-2.2毫米。该AP值前面的负号表示它是前囱为2.00mm后。对ML值之前的±符号表示从中心左，右方向。最后，将DV前的负号表示它是从大脑表面腹侧移动。 </li></ol> 4.立体框架安装<ol><li>佩戴外科口罩，无尘实验室外套，和无菌手术手套。 </li><li>擦拭立体定位仪和鼠标适配器，用70％的乙醇。 </li><li>躺在柜台无菌手术铺巾。 </li><li>放置在立体定位仪上的无菌手术单上面鼠标适配器。 </li><li>擦拭鼠标加热垫，用70％的乙醇。定位加热垫在立体框架床。使用通用labora保守党标签磁带到磁带上下加热垫在鼠标适配器床。 <ol><li>根据制造商的指示装上加热垫，以温水再循环器泵。 </li><li>打开温水再循环器泵和设定温度至37℃，然后等待，直到它达到的温度。 </li></ol></li><li>通过拧到根据制造商的方向电动垂直轴注射贴上50微升汉密尔顿注射器用29克针到立体框架。 </li><li>打开珠灭菌，并等待，直到它达到制造商的预设温度。 注:这是用来消毒的动物之间的不锈钢仪表。这需要仪器20秒，珠灭菌接触。 </li><li>打开热板，并将其设定为42℃，顶上放置一个干净的空笼子。 </li><li>而汉密尔顿注射器固定在立体框架上使用的机动立体我njector绘制了Aβ1-42的解决方案。 注意:绘制了超过4微升溶液到注射器，然后用立体定位喷射器设置注入量。根据制造商的说明书进行操作的喷射器。 </li></ol> 5.动物的制备<ol><li>确定鼠标的重量用电子秤。 </li><li>通过注入腹腔（IP）麻醉小鼠氯胺酮/甲苯​​噻嗪鸡尾酒在100毫克/千克氯胺酮和10毫克/公斤甲苯噻嗪。由脚趾捏反射损耗监测麻醉深度。 </li><li>之后鼠标镇静采取理发器，刮胡子它的头，露出的皮肤在颅骨。 </li><li>将鼠标上的立体定位床的顶部的加热垫的顶部。 </li><li>用锅铲开嘴，把门牙牙齿镇守内线。固定突出件在鼠标的表面上。夹紧它轻轻放下，而不是太紧。 </li><li>颇似和灰的双边横杆耳朵进外耳道，以确保头。移动每个横梁，直到它击中头骨，然后转动螺丝将其锁定。 <ol><li>为了确保头部固定用你的食指轻轻向下推头。如果头部被正确地固定它不会发出或按下时移动。 </li></ol></li><li>滴一滴眼霜或眼滴的眼睛，让他们湿润。确保眼睛是整个手术过程中的湿润。 </li><li>消毒手术部位用消毒棉签聚维酮碘溶液适用于皮​​肤在颅骨，其次是70％的乙醇。执行交替优碘和70％乙醇清洗2次以上。 </li></ol> 6.手术和输液<ol><li>用手术刀做一个2-3毫米的切口，在头皮中线，然后用直细剪刀切口线延伸至1.0-1.5厘米，以暴露矢状缝颅骨，地标，前囟，和lambda■哪些立体定位坐标基础上的。使用微夹具，保持皮肤分开。 注:前囟门和lambda位置在小鼠大脑图谱"鼠脑立体定位坐标"14解释。 </li><li>取棉签，浸泡在无菌盐水，并用它来清洗颅骨。然后，用干净的干棉签干燥头骨。 </li><li>使用无菌细笔以纪念前囟门点。使用立体显微使得针的尖端刚好触及前囟点来定位Hamilton注射器。 <ol><li>记录的DV坐标。 </li><li>要检查头骨的AP轴是水平移动汉密尔顿注射器向后，使针尖触及的lambda点。 </li><li>记录的DV位置。确保DV坐标前囟和lambda是在0.5毫米。 注意:我们的目标是最小化前囟和lambda之间的DV差。 </li></ol></li><li>使用Micromanipulator到Hamilton注射器针尖返回到前囟点。 </li><li>记录起始位置AP。 </li><li>减去2.00毫米从起始坐标AP计算AP结束位置。 </li><li>使用显微重新定位汉密尔顿注射器针头到最后的AP坐标。 </li><li>记录当前坐标ML。 </li><li>计算左，右结束ML坐标加上或减去1.3毫米从起始ML坐标。 </li><li>使用显微汉密尔顿注射器针头移到要么结束ML坐标。 <ol><li>触针尖在两个结束ML坐标颅骨的表面上，并记录的DV坐标，并确保该值是在0.5毫米。 注意:我们的目标是最小化两个结束ML坐标之间的的DV差。 </li><li>而在一个结局协调拉动针上0.5-1厘米以上的头骨。以无菌细笔，以纪念在日点头骨的E面。重复的头骨对侧此步骤。 </li></ol></li><li>摆动汉密尔顿注射器明确的出路。 </li><li>附加0.8毫米钻头的钻。取的钻头用双手，该表用于稳定性的表面上设置弯管，并略定位钻头上方的左侧或右侧的ML坐标Sharpie笔点。激活钻，下钻尖到颅骨表面引入头骨一个洞。重复对侧此步骤。 注意:有些出血可能由新引进的孔出现。如果有出血然后用干净的干棉签轻拍血液。 </li><li>移动汉密尔顿注射器放回原位了无论是新推出的ML孔。降低汉密尔顿针刚刚过去的头骨。在这一点上要小心，不要穿刺大脑。为了确保针头已通过颅骨把你的食指，轻轻推针对头骨进行河畔Ë针不会退出孔。 </li><li>记录起始DV坐标。 </li><li>计算减去从起始坐标DV2.2毫米结束DV坐标。 </li><li>降低针下来到结束DV坐标。 </li><li>激活立体注射器泵将4微升的Aβ1-42的成齿状回以0.5微升/分的速率。 </li><li>当输液完成让针头留在原地另外的1分钟，以最小化的溶液回流出注射部位。 </li><li>移动针到大脑的另一侧，并重复步骤6.13-6.18。 </li></ol> 7.动物搬运和术后护理<ol><li>拧耳朵酒吧和面部/头套。从该装置中取出的小鼠。 </li><li>用学生的标准模式钳拉近头皮，然后用密封缝合伤口。 </li><li>注射1毫升无菌生理盐水为通过IP鼠标水化。 </li><li>在拍摄对象丁丙诺啡（0.1毫克/公斤）皮下注射，以减轻疼痛。 </li><li>保持鼠标在干净的空笼子上42℃的热板的顶部设置，直到它被唤醒，然后用充足的食物和水返回到其住房笼。 </li><li>通过皮下注射给药丁丙诺啡每12小时3天缓解疼痛。 </li></ol> 8.拆线<ol><li>麻醉小鼠氯胺酮/甲苯​​噻嗪鸡尾酒在100毫克/千克氯胺酮和10毫克/公斤xlyazine通过IP。注:这是手术后7-10天进行。 </li><li>用学生的标准模式镊子和直细剪刀去除缝线。 </li><li>注射1毫升无菌生理盐水为通过IP鼠标水化。 </li><li>保持小鼠在一个干净的空笼上42℃的热板的顶部设置，直到其被唤醒，然后将其与充足的食物和水返回到其壳体笼。 </li></ol> 9.动物牺牲和组织处理<ol><li>麻醉小鼠和p用4％多聚甲醛15 erfuse它，取出大脑，并处理它cryosectioning 16。 注:动物献祭的时间取决于实验者。然而，对于我们的研究中，我们选择了7和14天手术后。 </li></ol>

<ol>
	<li>Baleriola, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonally+Synthesized+ATF4+Transmits+a+Neurodegenerative+Signal+across+Brain+Regions.">Axonally Synthesized ATF4 Transmits a Neurodegenerative Signal across Brain Regions.</a> Cell. 158 (5), 1159-1172 (2014).</li><li>Haass, C., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+protein+oligomers+in+neurodegeneration:+lessons+from+the+Alzheimer's+amyloid+beta-peptide.+Nature+reviews.">Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide. Nature reviews.</a> Molecular cell biology. 8 (2), 101-112 (2007).</li><li>Knowles, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+p75+neurotrophin+receptor+promotes+amyloid-beta(1-42)-induced+neuritic+dystrophy+in+vitro+and+in+vivo.">The p75 neurotrophin receptor promotes amyloid-beta(1-42)-induced neuritic dystrophy in vitro and in vivo.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 29 (34), 10627-10637 (2009).</li><li>Troy, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Amyloid-induced+neuronal+apoptosis+requires+c-Jun+N-terminal+kinase+activation.">beta-Amyloid-induced neuronal apoptosis requires c-Jun N-terminal kinase activation.</a> Journal of neurochemistry. 77 (1), 157-164 (2001).</li><li>Crews, L., Rockenstein, E., Masliah, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APP+transgenic+modeling+of+Alzheimer's+disease:+mechanisms+of+neurodegeneration+and+aberrant+neurogenesis.">APP transgenic modeling of Alzheimer's disease: mechanisms of neurodegeneration and aberrant neurogenesis.</a> Brain structure &amp; function. 214 (2-3), 111-126 (2010).</li><li>Gotz, J., Ittner, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+Alzheimer's+disease+and+frontotemporal+dementia.">Animal models of Alzheimer's disease and frontotemporal dementia.</a> Nature reviews. Neuroscience. 9 (7), 532-544 (2008).</li><li>Calhoun, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron+loss+in+APP+transgenic+mice.">Neuron loss in APP transgenic mice.</a> Nature. 395 (6704), 755-756 (1998).</li><li>Oakley, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraneuronal+beta-amyloid+aggregates,+neurodegeneration,+and+neuron+loss+in+transgenic+mice+with+five+familial+Alzheimer's+disease+mutations:+potential+factors+in+amyloid+plaque+formation.">Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 26 (40), 10129-10140 (2006).</li><li>Jean, Y. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-2+is+essential+for+c-Jun+transcriptional+activation+and+Bim+induction+in+neuron+death.">Caspase-2 is essential for c-Jun transcriptional activation and Bim induction in neuron death.</a> The Biochemical journal. 455 (1), 15-25 (2013).</li><li>Sotthibundhu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-amyloid(1-42)+induces+neuronal+death+through+the+p75+neurotrophin+receptor.">Beta-amyloid(1-42) induces neuronal death through the p75 neurotrophin receptor.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 28 (15), 3941-3946 (2008).</li><li>Kar, S., Quirion, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+beta+peptides+and+central+cholinergic+neurons:+functional+interrelationship+and+relevance+to+Alzheimer's+disease+pathology.">Amyloid beta peptides and central cholinergic neurons: functional interrelationship and relevance to Alzheimer's disease pathology.</a> Progress in brain research. 145, 261-274 (2004).</li><li>Leranth, C., Frotscher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+septal+region+in+the+rat+brain:+cholinergic-GABAergic+interconnections+and+the+termination+of+hippocampo-septal+fibers.">Organization of the septal region in the rat brain: cholinergic-GABAergic interconnections and the termination of hippocampo-septal fibers.</a> The Journal of comparative neurology. 289 (2), 304-314 (1989).</li><li>Fa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+oligomeric+beta-amyloid+1-42+and+induction+of+synaptic+plasticity+impairment+on+hippocampal+slices.">Preparation of oligomeric beta-amyloid 1-42 and induction of synaptic plasticity impairment on hippocampal slices.</a> Journal of visualized experiments : JoVE. (41), (2010).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2001).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of visualized experiments : JoVE. (65), (2012).</li><li>Currle, D. S., Monuki, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash+freezing+and+cryosectioning+E12.5+mouse+brain.">Flash freezing and cryosectioning E12.5 mouse brain.</a> Journal of visualized experiments : JoVE. (4), (2007).</li><li>Jin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+amyloid+beta-protein+dimers+isolated+from+Alzheimer+cortex+directly+induce+Tau+hyperphosphorylation+and+neuritic+degeneration.">Soluble amyloid beta-protein dimers isolated from Alzheimer cortex directly induce Tau hyperphosphorylation and neuritic degeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (14), 5819-5824 (2011).</li><li>Lambert, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusible,+nonfibrillar+ligands+derived+from+Abeta1-42+are+potent+central+nervous+system+neurotoxins.">Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins.</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (11), 6448-6453 (1998).</li><li>Masters, C. L., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry+of+amyloid+beta-protein+and+amyloid+deposits+in+Alzheimer+disease.">Biochemistry of amyloid beta-protein and amyloid deposits in Alzheimer disease.</a> Cold Spring Harbor perspectives in medicine. 2 (6), a006262 (2012).</li><li>Chakrabarti, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+receptor+agonists+for+attenuation+of+neuroinflammation+and+neurodegeneration.">Estrogen receptor agonists for attenuation of neuroinflammation and neurodegeneration.</a> Brain research bulletin. 109C, 22-31 (2014).</li><li>Cirrito, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+assessment+of+brain+interstitial+fluid+with+microdialysis+reveals+plaque-associated+changes+in+amyloid-beta+metabolism+and+half-life.">In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-beta metabolism and half-life.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 23 (26), 8844-8853 (2003).</li><li>Puzzo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picomolar+amyloid-beta+positively+modulates+synaptic+plasticity+and+memory+in+hippocampus.">Picomolar amyloid-beta positively modulates synaptic plasticity and memory in hippocampus.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 28 (53), 14537-14545 (2008).</li><li>Puzzo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+amyloid-beta+is+necessary+for+hippocampal+synaptic+plasticity+and+memory.">Endogenous amyloid-beta is necessary for hippocampal synaptic plasticity and memory.</a> Annals of. 69 (5), 819-830 (2011).</li><li>Akpan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+of+caspase-9+inhibitor+reduces+caspase-6-dependent+axon/neuron+loss+and+improves+neurological+function+after+stroke.">Intranasal delivery of caspase-9 inhibitor reduces caspase-6-dependent axon/neuron loss and improves neurological function after stroke.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 31 (24), 8894-8904 (2011).</li><li>Fulmer, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-derived+BDNF+supports+myelin+protein+synthesis+after+cuprizone-induced+demyelination.">Astrocyte-derived BDNF supports myelin protein synthesis after cuprizone-induced demyelination.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 34 (24), 8186-8196 (2014).</li><li>Thakker-Varia, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuropeptide+VGF+is+reduced+in+human+bipolar+postmortem+brain+and+contributes+to+some+of+the+behavioral+and+molecular+effects+of+lithium.">The neuropeptide VGF is reduced in human bipolar postmortem brain and contributes to some of the behavioral and molecular effects of lithium.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 30 (28), 9368-9380 (2010).</li><li>Greferath, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enlarged+cholinergic+forebrain+neurons+and+improved+spatial+learning+in+p75+knockout+mice.">Enlarged cholinergic forebrain neurons and improved spatial learning in p75 knockout mice.</a> The European journal of neuroscience. 12 (3), 885-893 (2000).</li><li>Brashear, H. R., Zaborszky, L., Heimer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+GABAergic+and+cholinergic+neurons+in+the+rat+diagonal+band.">Distribution of GABAergic and cholinergic neurons in the rat diagonal band.</a> Neuroscience. 17 (2), 439-451 (1986).</li><li>Clarke, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+innervation+of+the+rat+dentate+gyrus:+an+immunocytochemical+and+electron+microscopical+study.">Cholinergic innervation of the rat dentate gyrus: an immunocytochemical and electron microscopical study.</a> Brain research. 360 (1-2), 349-354 (1985).</li><li>Garcia-Osta, A., Alberini, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+beta+mediates+memory+formation.">Amyloid beta mediates memory formation.</a> Learning &amp; memory. 16 (4), 267-272 (2009).</li></ol>

作者宣称，他们有没有竞争的财务权益。

为了实现成功的Aβ1-42注射实验者或外科医生必须:1）使用无菌技术; 2）正确识别的准确坐标兴趣大脑区域; 3）能够正确地固定在鼠标的立体框架与夷为平地，在​​AP和ML轴的大脑; 4）必须与精密操作的微操作的能力; 5）确保适当的术后护理。如果遵循这些关键步骤，鼠标应该生存下去，没有观察到感染的手术。 符合体外研究中 ，我们和其他人已经表明，Aβ1-42直接注入引起海马神经元死亡1,4,9,10，描绘了AD的主要特点之一。变性是在注射部位的附近显而易见所示由一个在被补充增加标记变性1齿状回的体积减小。此外，T他胆碱投射到海马神经元死在逆行方式如图中的胆碱能标记的减少，并在高炉1,10增加TUNEL阳性染色。因此，这种体内模型可作为一个很好的模式在当地进行调查Aβ1-42的半急性作用。该注射模式的主要优点是它的动态性:任何大脑区域可能是针对不同年龄的动物都可以使用。 9个月的雄性小鼠，选择在我们的实验中，因为我们相信越来越多的Aβ1-42各级老年小鼠更好地模拟发生在老年人的疾病。此外，我们的目标是保持该实验通过使用相同年龄的小鼠一致。但是，也可以用于注射任何年龄的小鼠。在过去，我们已经尝试上为16个月大的老鼠和其他人使用的是2个月大的小鼠9,10。只有雄性小鼠应该研究，作为雌性小鼠表现出ESTRO使用根水平波动和雌激素已知具有神经保护作用20。虽然许多研究兴趣周围的Aβ1-42的病理作用中心，这里也有证据表明，低或生理的Aβ1-42水平是必需的正常功能的学习和记忆21,22。在这些研究中调查降低的Aβ1-42的浓度注入，注入成海马22,23，有效地揭示了这种模式的效用的动态特性的另一个例子。已成功地注入立体定位的其它化合物包括药物，病毒，的siRNA，抗体，和肽1,22-26调查各种效果，从细胞死亡/存活动物的行为。因此，有许多应用程序使用此输液范例。 所描述的方法输液，同时表现出不同的潜力以V龙腾光电研究使用这种技术，还带有固有的局限性。第一，这种模式只尝试重现分离的大脑区域的Aβ1-42的影响。其次，注射部位被从引入针的进入脑损伤。这有助于额外的细胞死亡和神经胶质增生。因此，分析已被从喷射道执行程。与同大脑对侧相应的车辆控制这不应该是一个问题，因为注入的车辆和Aβ1-42的解决方案蔓延到周围的组织。第三，因为高炉胆碱能神经元投射到海马的海马如显然由DG的由针（ 图1B）破裂时的物理损坏可能会无意中杀死一些BFCNs。幸运的是，检查神经回路在短期内 - 1周单次输注的内 - 不因为我们的数据表明，增加构成问题胆碱能死亡在基底前脑中是Aβ1-42的结果，而不是从针的创伤;注射控制提供相应的分析。这些数据表明，对DG的退化也是必要的高炉胆碱能神经元的损失，这些神经元丧失其突触目标（ 图2B）。不幸的是，对动物存活长达2周的注射媒介物的侧示BFCN死亡显著海拔1周以上后车辆注射的动物似乎是部分由于从的物理创伤引起的齿状回的变性和减薄针。然而，即使在此阶段中的数据表明，有一个在高炉同侧的Aβ1-42-injected站点显著更多的死亡。第四，识别细微的解剖边界可能难以实现。例如，中隔和对角带之间的边界的基础上的前的位置被确定合缝，这种技术是先前描述的27。此外，由于同侧的BF胆碱能神经元投射的-主要是从内侧隔核和斜角带（MS / DB）的垂直肢-海马12,28,29的齿状回我们决定注入一个半球与Aβ 1-42，并使用相对半球作为对照进行比较。可以想象的是，对于这个范例的关注将是正确的标识分离左侧和右侧的MS / DB中的边界。而该MS是在中线区域时，DB是多个横向。对于这个目前的工作和我们最近的出版物1，我们能够分辨舒适左右DB。我们的定量揭示的ChAT（+）神经元减少了约25％的在DB中的Aβ1-42-injected侧。然而，我们没有检测到在MS 1中的ChAT的任何显著改变（+）神经元。由于横向的禄DB的通货膨胀和观察到的变化我们认为我们有理由相同的动物中使用的车辆和Aβ1-42注射。此外，相同的动物中检测2个不同的实验条件下，不仅最大限度地利用的动物，但也减少了潜在的动物到动物可变性。而我们的实验设计可以让我们比较左和右半球，可以想象该比较技术可能不是在今后的研究， 也就是可行的，如果中隔是研究的主要焦点。在这种情况下，每个动物将必须作为一个实验条件。因此，使用动物是在实验者的判断。第五，可以在电流注入模型可用于其它读奏如行为？我们在使用电流的Aβ1-42注射模型目的是评估的Aβ1-42的神经病理学作用在体内 ，并将其与在体外的研究结果1 </suP&gt;。为此目的，它提供了目的。 AD是一种疾病，影响个体的记忆功能和行为功能。因此，自然要问当前的模式是否可以被用于研究的行为。扩大现有的模式进入行为研究实验者将不得不模型进一步刻画，可能要调整喷油策略。例如，因为我们观察到DG变性（ 图1B和2B）时，我们的目标注射针进入或接近DG在第7天对DG的破坏可能产生意想不到的行为异常。因此，如果一个研究需要检查的Aβ1-42对海马依赖性行为的影响，则注入目标可能必须海马邻近直接进入它重新定位，而不是和允许的Aβ1-42扩散到海马。这就是说，利用电流注入范例行为的测试是可能的，但需要更多的测试ING和表征，以及最终的实验策略将由终端用户确定。有趣的是，已经有涉及外部传递Aβ1-42直接进入大脑前啮齿类动物行为的研究。22,30总之，这些限制需要在小鼠体内研究采用这种模型设计时要考虑的。 由于存在没有单一的小鼠模型捕捉广告的完整病理作用的立体Aβ1-42输液技术提供实验者在体内 Aβ1-42小鼠模型的替代。当由训练有素的个人进行该模型是最适合于短期研究重点对Aβ1-42的一个特定的大脑区域或电路的影响。

淀粉样蛋白斑沉积，这是由淀粉样蛋白-β（Aβ1-42）的，是阿尔茨海默氏病（AD）的病理学的关键特征。大量研究表明，过高或毒性水平的重组低聚Aβ1-42引起神经元死亡，突触营养不良，损失和功能障碍;以及学习记忆障碍1-4。受影响的脑区域包括海马，皮质，并如基底前脑和扁桃体5,6-皮层下结构。迄今为止，有试图模拟的Aβ1-42病理学的AD的多个转基因小鼠模型。根据应变这些动物被证明是在审查AD的选择病理特征非常有用。不幸的是，除2转基因品系，APP23和5XFAD，这些小鼠从未完全复制的神经元损失，AD的一个关键方面。即使在APP23和5XFAD观察神经元的损失，神经元死亡，安装前后VED是微妙的，年龄依赖性，孤立的几个选择地区7,8。 直接注入低聚的Aβ1-42入野生型小鼠的大脑提供的体内模型它复制amyloidopathy 1,9,10的神经元死亡方面极好。不像通常用于转基因小鼠模型的低聚的Aβ1-42输注模型是理想的急性升降的Aβ1-42的水平在空间和时间的方式。使用野生型小鼠对这种模式的优点避免从在转基因小鼠系引入的突变势补偿或副作用。过去的研究已经表明，注入的Aβ1-42的毒性水平进海马引起神经元死亡在注射部位的附近1周1次内。此外，与观察一致，即Aβ1-42为胆碱能神经元11的基底前脑的有毒胆碱能神经元（BFCN）人群中伸出的海马在7-14天以下的β-淀粉样蛋白注射1,10小鼠下降20-50％，从而有效的隔离神经回路中的脑组织考试。因为BFCN项目同侧海马12的齿状回，在大多数情况下控制/车辆和低聚的Aβ1-42溶液可对大脑允许将左和右半球1之间进行比较的任一侧被注入。 在这份报告中，我们将提供成人野生型C57BL / 6J小鼠进行详细的手术和注射方法。这种小鼠品系被选中，是因为它在研究广泛应用。从技术上讲，任何脑区域可以针对输液，但在这里，我们将使用海马的齿状回作为目标来说明该技术。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ketamine HCl (100mg/ml)</td>
			<td>Henry Schein Medical</td>
			<td>1049007</td>
			<td>100 mg ketamine per 1 kg animal</td>
		</tr>
		<tr>
			<td>Xylazine (20mg/ml)</td>
			<td>Henry Schein Medical</td>
			<td>not available</td>
			<td>10 mg xylazine per 1 kg animal</td>
		</tr>
		<tr>
			<td>Buprenex (0.3mg/ml)</td>
			<td>Henry Schein Medical</td>
			<td>1217793</td>
			<td>0.1 mg buprenex per 1 kg animal</td>
		</tr>
		<tr>
			<td>A&#946;1-42</td>
			<td>David Teplow/UCLA</td>
			<td>not available</td>
			<td>100 &#956;M; This amyloid was used in the paper</td>
		</tr>
		<tr>
			<td>A&#946;1-42</td>
			<td>Bachem</td>
			<td>H-1368</td>
			<td>Can be used in place of amyloid from the Teplow lab</td>
		</tr>
		<tr>
			<td>A&#946;1-42</td>
			<td>American Peptide</td>
			<td>62-0-80B</td>
			<td>Can be used in place of amyloid from the Teplow lab</td>
		</tr>
		<tr>
			<td>Scrambled A&#946;1-42</td>
			<td>American Peptide</td>
			<td>62-0-46B</td>
			<td>Can be used as control peptide for comparing A&#946;1-42</td>
		</tr>
		<tr>
			<td>NU4 Antibody (Oligomeric Amyloid Antibody)</td>
			<td>Gift from William Klein/Northwestern U.</td>
			<td>not available</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>Anti-Amyloid Oligomeric Antibody&#160; (Polyclonal Rabbit)</td>
			<td>EMD Millipore</td>
			<td>AB9234</td>
			<td>May be used in place of Nu4; needs to&#160; be tested by the end user</td>
		</tr>
		<tr>
			<td>6E10 Antibody (Monoclonal Mouse) (Amyloid Antibody)</td>
			<td>Biolegend</td>
			<td>sig-39320</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>ChAT Antibody (Polyclonal Goat)</td>
			<td>Millipore</td>
			<td>AB144P</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>DeadEnd Fluorometric TUNEL system</td>
			<td>Promega</td>
			<td>G3250</td>
			<td>Follow manufacturer&#39;s directions for use</td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td>Use when coverslipping slides</td>
		</tr>
		<tr>
			<td>Fluorogold</td>
			<td>Fluorochrome, LLC</td>
			<td>not available</td>
			<td>2% solution</td>
		</tr>
		<tr>
			<td>Absolute Ethanol (200 proof)</td>
			<td>Fisher Scientific</td>
			<td>BP2818-4</td>
			<td>For making 70% ethanol for sanitizing and disinfecting</td>
		</tr>
		<tr>
			<td>Novex 10-20% Tricine gel</td>
			<td>Life Technologies</td>
			<td>EC6625BOX</td>
			<td>For separating A&#946;1-42</td>
		</tr>
		<tr>
			<td>Novex Tricine SDS Running Buffer (10X)</td>
			<td>Life Technologies</td>
			<td>LC1675</td>
			<td>For running 10-20% Tricine gels</td>
		</tr>
		<tr>
			<td>Novex Tris-Glycine Transfer Buffer (25X)</td>
			<td>Life Technologies</td>
			<td>LC3675</td>
			<td>For transferring 10-20% Tricine gels</td>
		</tr>
		<tr>
			<td>SuperSignal Western Blot Enhancer</td>
			<td>Thermo Scientific</td>
			<td>46640</td>
			<td>For enhancing A&#946;1-42 signal; follow manufacturer&#39;s protocol</td>
		</tr>
		<tr>
			<td>Protran BA79 Nitrocellulose Blotting Membrane, 0.1 &#956;m</td>
			<td>GE Healthcare Life Sciences</td>
			<td>10402088</td>
			<td>For transferring 10-20% Tricine gels</td>
		</tr>
		<tr>
			<td>Xcell SureLock Mini-Cell</td>
			<td>Life Technologies</td>
			<td>EI0001</td>
			<td>Electrophoresis aparatus for running 10-20% Tricine gels</td>
		</tr>
		<tr>
			<td>GenTeal Lubricant Eye Gel</td>
			<td>Novartis</td>
			<td>not available</td>
			<td>For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores</td>
		</tr>
		<tr>
			<td>Refresh Optive Lubricant Eye Drops</td>
			<td>Allergan</td>
			<td>not available</td>
			<td>For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores; Can be used in place of GenTeal</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Stoelting</td>
			<td>50998</td>
			<td>For sanitizing and disinfecting</td>
		</tr>
		<tr>
			<td>Round/Tapered Spatula&#160;</td>
			<td>VWR</td>
			<td>82027-490</td>
			<td>For opening animal mouth</td>
		</tr>
		<tr>
			<td>Bulldog Serrefines Clamps (Jaw Dims. 9X1.6mm; Length 28mm)</td>
			<td>Fine Science Tools</td>
			<td>18050-28</td>
			<td>Optional; For keeping scalp skin apart during injection</td>
		</tr>
		<tr>
			<td>Straight Fine Scissors (Cutting edge 25mm; Length 11.5cm)</td>
			<td>Fine Science Tools</td>
			<td>14060-11</td>
			<td>For cutting scalp</td>
		</tr>
		<tr>
			<td>#3 Scalpel Handle</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>#11 Surgical Blade</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td>For making scalp incision</td>
		</tr>
		<tr>
			<td>Student Standard Pattern Forcep (Tip Dims. 2.5x1.5mm; Length 11.5cm)</td>
			<td>Fine Science Tools</td>
			<td>91100-12</td>
			<td>For holding scalp closed during suturing</td>
		</tr>
		<tr>
			<td>Trimmer Combo Kit</td>
			<td>Kent Scientific</td>
			<td>CL9990-1201</td>
			<td>For shaving hair</td>
		</tr>
		<tr>
			<td>T/Pump Warm Water Recirculator&#160;</td>
			<td>Kent Scientific&#160;</td>
			<td>TP-700</td>
			<td>For warming animal during surgery</td>
		</tr>
		<tr>
			<td>Resusable Warmining Pad (5&#34; x 10&#34;)</td>
			<td>Kent Scientific&#160;</td>
			<td>TPZ-0510FEA</td>
			<td>For attaching it to the T/Pump warm water recirculator to warm the animal during surgery</td>
		</tr>
		<tr>
			<td>Cordless Micro Drill</td>
			<td>Stoelting</td>
			<td>58610</td>
			<td>Use 0.8mm steel burrs to drill holes in the skull</td>
		</tr>
		<tr>
			<td>Lab Standard Stereotaxic Instrument with Mouse &#38; Neonatal Rat Adaptor</td>
			<td>Stoelting</td>
			<td>51615</td>
			<td></td>
		</tr>
		<tr>
			<td>Just for Mouse Stereotaxic Instrument</td>
			<td>Stoelting</td>
			<td>51730</td>
			<td>Can use this in place of Stoelting Cat. #51615</td>
		</tr>
		<tr>
			<td>Quintessential Stereotaxic Injector</td>
			<td>Stoelting</td>
			<td>53311</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Glass Bead Sterilizer</td>
			<td>Stoelting</td>
			<td>50287</td>
			<td>For sterilizing stainless steel instruments</td>
		</tr>
		<tr>
			<td>Sterile Surgical Drape (18&#34; x 26&#34;)</td>
			<td>Stoelting</td>
			<td>50981</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton Syringe 50 ml, Model 705 RN SYR, NDL</td>
			<td>Hamilton Company</td>
			<td>7637-01</td>
			<td>For brain injection; use different syringes for different solutions</td>
		</tr>
		<tr>
			<td>29 Gauge Needle, Small Hub RN NDL</td>
			<td>Hamilton Company</td>
			<td>7803-06</td>
			<td>For attaching to the Hamilton syringe for brain injection</td>
		</tr>
		<tr>
			<td>1 ml BD Tuberculin Syringes</td>
			<td>VWR</td>
			<td>BD309659</td>
			<td>For administering anesthesia and saline</td>
		</tr>
		<tr>
			<td>30 Gauge Needle (0.5&#34;)</td>
			<td>VWR</td>
			<td>BD305106</td>
			<td>For administering anesthesia and saline</td>
		</tr>
		<tr>
			<td>Portable Electronic CS Series Scale (Ohaus)</td>
			<td>VWR</td>
			<td>65500-202</td>
			<td>For weighing animals to determine anesthesia dose</td>
		</tr>
		<tr>
			<td>Hot plate (Top Plate Dims. 7.25x7.25in)</td>
			<td>VWR</td>
			<td>47751-148</td>
			<td>For warming animals post-surgery</td>
		</tr>
		<tr>
			<td>Sofsilk Silk Suture C-1 Cutting 3/8, 12 mm</td>
			<td>Covidien</td>
			<td>S1173</td>
			<td>For closing wound</td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhesive (3M)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-361931</td>
			<td>Optional: for aiding in wound closure; Use with suture.</td>
		</tr>
		<tr>
			<td>Cotton-Tipped Wooden-Shaft Sterile Applicators</td>
			<td>Fisher scientific</td>
			<td>22-029-488</td>
			<td>For cleaning and drying surgical wound</td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific&#160;</td>
			<td>12-550-15</td>
			<td>For collecting brain sections</td>
		</tr>
		<tr>
			<td>VWR Micro Cover Glass 24 X 50 mm</td>
			<td>VWR</td>
			<td>48393241</td>
			<td>For mounting microscope slides</td>
		</tr>
		<tr>
			<td>Thermo Scientific Nalgene Syringe Filter 0.2 &#956;m</td>
			<td>Fisher Scientific</td>
			<td>194-2520</td>
			<td>For sterilizing saline solution</td>
		</tr>
		<tr>
			<td>Sterile dual tip skin markers by Viscot Medical</td>
			<td>Medline</td>
			<td>VIS1422SRL91</td>
			<td>For marking coordinates on the skull</td>
		</tr>
	</tbody>
</table>

stereotaxic infusion of oligomeric amyloid-beta into the mouse hippocampus

阿尔茨海默氏病是一种神经退行性疾病，影响人口老化。该疾病的一个主要神经病理学特征是过度产生淀粉样蛋白β的和的β-淀粉样斑块中的患病个体的脑区的沉积。多年来科学家已经产生的试图复制的淀粉样蛋白β病理学众多阿尔茨海默氏病小鼠模型。不幸的是，在小鼠模型中只选择性地模仿疾病特征。神经元死亡，在早老性痴呆病患者的脑中一个显着的效果，是明显缺乏在这些小鼠。因此，我们和其他人已采用直接注入淀粉样蛋白β的可溶性低聚物质的方法 - 淀粉样蛋白β的已被证明是最毒的神经元的形式 - 立体定向到大脑。在本报告中，我们利用雄性C57BL / 6J小鼠文件增加一个选择脑区域淀粉样蛋白β水平的这种手术技术。该输液目标是海马的齿状回，因为这大脑结构，沿与由胆碱能电路连接在所述基底前脑，表示的变性疾病的领域之一。提升β​​-淀粉样蛋白在通过立体输液齿状回的结果显示增加神经元丢失在齿状回1周内，虽然在布罗卡的斜角带垂直肢体同时增加细胞死亡和胆碱能神经元缺失的基底前脑。这些影响观察到2周。我们的数据表明，目前的β-淀粉样蛋白输注模型提供了一种替代的小鼠模型在一个短期的基础上处理区域特定神经元死亡。该模型的优点在于，β淀粉样蛋白可在空间和时间的方式升高。

制备人重组低聚Aβ1-42的本发明的方法产生的可溶性低聚物质选自单体，二聚体，三聚体和四聚体（ 图1A）的。这些低分子量的Aβ1-42物种，而不是原纤维和斑块，已被证明在许多设置是最毒的神经元1,4,9,17-19。以确定是否低聚的Aβ1-42诱导的神经元死亡的鼠脑的Aβ1-42（4微升100μM的原液）被注入海马DG在9个月大的野生型C57BL一个半球/ 6J小鼠。一个海马为C57BL / 6J的重量估计为围绕0.018克和因此的Aβ1-42的递送约100微克/克。相同的鼠标大脑对侧DG被注入了一种控制/车辆解决方案-其中包括二甲基亚砜溶解Aβ1-使用等量42稀释在（1×）的PBS -用于比较。如前面提到的加扰或反向的Aβ1-42肽可以用作控制进行比较，因为我们先前发现对海马神经元1没有影响。引进的针入脑的创建局部组织损伤（ 图1B），并导致最终DG崩溃我们选择来执行后喷射分析相邻的喷射道，其中所述组织是仍然完好无损。 1周以内的Aβ1-42注射的DG呈现升高的Aβ1-42的水平的分子层，并在注射部位（ 图2A）的附近的多态细胞层。在7天和14天的Aβ1-42引起增加TUNEL阳性染色及减少的DG厚度，确认的Aβ1-42（ 图2B）的神经变性的影响。 因为高炉胆碱能神经元投射到DG海马我们确定下一如果在DG的神经元丢失触发BF变性。在这项研究中逆行示踪剂荧光金（2％悬浮液）是联合注射Aβ1-42到DG沿。后7天注射荧光金是在高炉（ 图2C），表明该标记物通过胆碱能神经元逆行运输检测。因此，对于量化在高炉的目的，我们选择了荧光金阳性细胞，因为这表明神经元端子暴露于的Aβ1-42在DG。在这两个1和2周后DGAβ1-42输液，斜角带的核心-对BF的一个分区，其中胆碱能神经元投射到DG -同侧Aβ1-42 -injected现场展出的增加TUNEL染色及减少的胆碱能神经元（ 图2D）。在高炉中观察到的神经元丢失证实过去的研究联系起来的破坏性Aβ1-42效应的BF-海马通路1,10。它也是至关重要的注意，从2周时间点的1周的时间点上对角带，其中，控制/车辆溶液注射在DG有进一步增加TUNEL染色和核的一侧的比较降低在胆碱能神经元（ 图2D，表）。这些结果反映在GCL厚度在2后喷射的时间点之间比较车辆注射侧的DG TUNEL阳性标记（ 图2B，表）显著减少和增加，这意味着引起针的物理创伤有助于变性随着时间的推移。甚至与针的这种不希望有的副作用，的Aβ1-42的神经变性效果仍然显著大于对照/载体溶液（ 图2D）的。两者合计，电流输注模型effectivelŸ再现Aβ1-42在小鼠大脑的毒性作用于7天内使之成为一个有吸引力的模型来研究短期Aβ1-42刺激。 <img alt="图1" src="/files/ftp_upload/52805/52805fig1.jpg" /> 图1.低聚的Aβ1-42制备和小鼠大脑注射道可视化。（A）的 2μM的低聚的Aβ1-42上分离10-20％甘氨酸凝胶，并转移到硝酸纤维素膜上。将膜印迹用的Aβ1-42抗体6E10（1:1000）。 （二）将Aβ1-42（4微升100μM的溶液）或对照/车辆输注9个月龄C57BL / 6J小鼠的成海马的左边和右边的DG分别。小鼠存活7天。脑被连续切片在每节20微米。海马切片进行DAPI染色。哈希标记描述注射道。箭头显示倒塌DG。比例尺代表200微米。 <a href="https://www.jove.com/files/ftp_upload/52805/52805fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52805/52805fig2.jpg" /> 图2.低聚的Aβ1-42诱导的神经元死亡中的DG和高炉。控制/车辆或Aβ1-42（4微升100μM的溶液）注入海马的左边和右边的DG分别为9月龄的C57BL / 6J小鼠。小鼠存活7或14天。脑被连续切片在每节20微米。 （A）后7天注射海马切片染色Aβ1-42（NU4抗体，1:2000）。 ML，分子层; GCL，神经节细胞层; PCL，多形细胞层。比例尺条为50微米。 （B </str翁&gt;）7或14天后染色DAPI或注射TUNEL海马部分。 * P &lt;0.05相对于1周。 *** P &lt;0.001相对于1周。 * P &lt;0.05相对于车辆。 ** P &lt;0.01相对于车辆。比例尺条为50微米。 （每组5只小鼠）（C）的车辆或Aβ1-42聚合共注射荧光金（2％溶液）。在高炉后7天注射荧光金标记来确定。含BF载玻片随机选择。荧光金标记被用来确定感兴趣区域。一旦感兴趣区域被确定邻近脑切片用于染色用于感兴趣的标记。的插图是其中在（D）中的图像被选择的区域。比例尺条为200微米。 （D）的 7天或14天后染色的ChAT注入BF部分（1:100，山羊多克隆）或TUNEL。 *** P &lt;0.001相对于1周。 * P &lt;0.05相对于车辆。定量分析对整个区域进行感兴趣。中隔和对角带之间的边界是基于前连合的位置描绘。的比例尺条为100μm。 （N = 5只小鼠）。 <a href="https://www.jove.com/files/ftp_upload/52805/52805fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus at JoVE.com

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

低聚淀粉样蛋白-β的立体灌注到小鼠海马

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

stereotaxic-infusion-oligomeric-amyloid-beta-into-mouse

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

18.0K 次观看.  Columbia University Medical Center. Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

视频: Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

超声引导下显微注射到小鼠前脑在子宫内在E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

科研

神经科学

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

特定脑区的兴奋毒性损伤的成年大鼠立体定向手术治疗

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

立体定向注射病毒载体的基因操作在小鼠脊髓条件

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Building An Open-source Robotic Stereotaxic Instrument

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for around $1,000 (excluding a drill), using industry standard stepper motors and CNC controlling software. Each axis has variable speed control and may be operated simultaneously or independently. The robot&#39;s flexibility and open coding system (g-code) make it capable of performing custom tasks that are not supported by commercial systems. Its applications include, but are not limited to, drilling holes, sharp edge craniotomies, skull thinning, and lowering electrodes or cannula. In order to expedite the writing of g-coding for simple surgeries, we have developed custom scripts that allow individuals to design a surgery with no knowledge of programming. However, for users to get the most out of the motorized stereotax, it would be beneficial to be knowledgeable in mathematical programming and G-Coding (simple programming for CNC machining).
The recommended drill speed is greater than 40,000 rpm. The stepper motor resolution is 1.8&#176;/Step, geared to 0.346&#176;/Step. A standard stereotax has a resolution of 2.88 &#956;m/step. The maximum recommended cutting speed is 500 &#956;m/sec. The maximum recommended jogging speed is 3,500 &#956;m/sec. The maximum recommended drill bit size is HP 2.

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (computer numeric controlled; CNC) stereotaxic instrument for around $1,000 (excluding a drill).

建设一个开放源码的机器人立体定位仪

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

鼠标显微注水技术进行有针对性的物质传递到中枢神经系统<i&gt;通过</i&gt;的颈内动脉

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

新生小鼠脑巴细胞基因调控的立体定向手术

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

行为小鼠海马空间受限振荡的记录

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

将化学遗传工程皮质内神经内生前生移植到早期产后小鼠大脑

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the neurosciences. Phylogenetically, these animals are much closer to humans than rodents. They also display complex behaviors, including a wide range of vocalizations and social interactions. Here, an effective stereotaxic neurosurgical procedure for implantation of recording electrode arrays in the common marmoset is described. This protocol also details the pre- and postoperative steps of animal care that are required to successfully perform such a surgery. Finally, this protocol shows an example of local field potential and spike activity recordings in a freely behaving marmoset 1 week after the surgical procedure. Overall, this method provides an opportunity to study the brain function in awake and freely behaving marmosets. The same protocol can be readily used by researchers working with other small primates. In addition, it can be easily modified to allow other studies requiring implants, such as stimulating electrodes, microinjections, implantation of optrodes or guide cannulas, or ablation of discrete tissue regions.

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset Callithrix jacchus

This work presents a protocol to perform a stereotaxic, neurosurgical implantation of&#160;microelectrode arrays in the common marmoset. This method specifically enables electrophysiological recordings in freely behaving animals but can be easily adapted to any other similar neurosurgical intervention in this species (e.g., cannula for drug administration or electrodes for brain stimulation).