我们感谢她的解剖技术和支持她的电影的女士亚希藤田保健大学宫川指令京都大学博士洋子锅岛。这项工作是促进基础研究的国家生物医药创新，为科学研究的优先领域，结合脑研究（师恩）赠款援助研究所健康科学计划 - 从日本文部科学省和支持一个格兰特 - 日本科学技术振兴机构佳洁士援助。

海马齿状回的剖析<ol><li>在深度麻醉的鼠标，仔细解剖大脑从头骨，放入冰冷的磷酸盐缓冲液（PBS）。 </li><li>含有冰冷的PBS在培养皿菜，切沿大脑纵裂的大脑使用手术刀，切断后的lambda（中脑，后脑和小脑）的地区。 </li><li>将大脑半球的内侧，使用镊子，小心地取出一个夹层显微镜下的间脑（丘脑和下丘脑）。这将会使内侧的海马，齿状回的可视化。齿状回阿蒙的角分辨它们之间的差距。海马损伤或周边地区，将使其更难以隔离齿状回。 </li><li>插入到的齿状回（在齿状回和阿蒙的角界限;图1）两侧的一个尖锐的针，针尖（例如，27 - 压力表针），和幻灯片沿的海马septo的时空轴针表面隔离齿状回。 </li><li>拿起孤立的齿状回用针或镊子，并放置在一个样品管。因此获得的齿状回组织样本可立即使用任何检测或存储在深冷冻供日后使用。 </li><li>隔离用同样的方法从其他脑半球的齿状回。 </li></ol>实时定量PCR 齿状回分离使用上述的方法，其余海马解剖阿蒙的从野生型小鼠的号角样本。 β-肌动蛋白，TDO2，DSP Mrg1b和Tyro3实时PCR技术进行了齿状回和阿蒙的号角样本描述以前1。引物5&#39; - CTGGCGAGATCACGATGACG和5&#39; - AAGCTACGCTGTTGTCTAACC用于Mrg1b，GCCTCCAAATTGCCCGTCA和5&#39; - CCAGCACTGGTACATGAGATCA Tyro3。 微阵列分析微阵列实验与男性的野生型小鼠和小鼠钙/钙调蛋白依赖性蛋白激酶Ⅱα-异构体的杂合子（α- CaMKII + / -小鼠）以前1。简单地说，从整个海马齿状回或野生型和突变型小鼠中分离出的RNA杂交小鼠基因组430 2.0阵列（Affymetrix公司，美国加州圣克拉拉市），每个基因芯片是由Affymetrix基因芯片扫描仪3000（GCS3000）扫描。基因芯片与芯片分析套件5.0版进行分析。

<ol>
	<li>Yamasaki, N., Maekawa, M., Kobayashi, K., Kajii, Y., Maeda, J., Soma, M., Takao, K., Tanda, K., Ohira, K., Toyama, K., Kanzaki, K., Fukunaga, K., Sudo, Y., Ichinose, H., Ikeda, M., Iwata, N., Ozaki, N., Suzuki, H., Higuchi, M., Suhara, T., Yuasa, S., Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-CaMKII+deficiency+causes+immature+dentate+gyrus,+a+novel+candidate+endophenotype+of+psychiatric+disorders.">Alpha-CaMKII deficiency causes immature dentate gyrus, a novel candidate endophenotype of psychiatric disorders.</a> Mol. Brain. 1, 6-6 (2008).</li><li>Insausti, A. M., Megias, M., Crespo, D., Cruz-Orive, L. M., Dierssen, M., Vallina, I. F., Insausti, R., Florez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+volume+and+neuronal+number+in+Ts65Dn+mice:+a+murine+model+of+Down+syndrome.">Hippocampal volume and neuronal number in Ts65Dn mice: a murine model of Down syndrome.</a> Neurosci. Lett. 253, 175-175 (1998).</li><li>Redwine, J. M., Kosofsky, B., Jacobs, R. E., Games, D., Reilly, J. F., Morrison, J. H., Young, W. G., Bloom, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dentate+gyrus+volume+is+reduced+before+onset+of+plaque+formation+in+PDAPP+mice:+a+magnetic+resonance+microscopy+and+stereologic+analysis.">Dentate gyrus volume is reduced before onset of plaque formation in PDAPP mice: a magnetic resonance microscopy and stereologic analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 1381-1381 (2003).</li><li>Lein, E. S., Zhao, X., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+molecular+atlas+of+the+hippocampus+using+DNA+microarrays+and+high-throughput+in+situ+hybridization.">Defining a molecular atlas of the hippocampus using DNA microarrays and high-throughput in situ hybridization.</a> J. Neurosci. 24, 3879-3879 (2004).</li><li>Matsuo, N., Yamasaki, N., Ohira, K., Takao, K., Toyama, K., Eguchi, M., Yamaguchi, S., Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+activity+changes+underlying+the+working+memory+deficit+in+alpha-CaMKII+heterozygous+knockout+mice.">Neural activity changes underlying the working memory deficit in alpha-CaMKII heterozygous knockout mice.</a> Front. Behav. Neurosci. 3, 20-20 (2009).</li><li>Franklin, K. B. J., Paxinos, G. <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. , (1997).</li></ol>

按照实验动物护理和使用指南“国家卫生研究所进行了动物实验，和藤田保健大学动物护理和使用委员会的批准。

齿状回海马2,3卷中占有约25％至30％ 。它具有独特的细胞组成，并在不同的大脑功能起着至关重要的作用。因此，隔离齿状回的技术分析特别是在这一地区发生的事件非常有用。 在这里，我们展示了一个过程，有效地解剖从成年小鼠海马齿状回，并确认该技术的精度。首先，组织学研究表明，齿状回分离没有其他地区的污染（图1），这表明可以准备一个纯粹的齿状回样本。 其次，我们确认解剖组织齿状回进行实时PCR齿状回特定的基因，TDO2和DSP，并亚扪人的号角丰富的基因，Mrg1b Tyro3 4（图2） 。对TDO2的基因表达式（p = 0.000023; n的分别= 4）和DSP（p = 0.0000030; n的分别= 4）的齿状回的样品被检测到明显较高的水平，而Mrg1b（p = 0.000080，N = 4和4）和Tyro3（P = 0.00017; n的= 4和4）分别较低的水平，比那些在阿蒙的号角样本。 β-肌动蛋白的表达水平没有差异，在这些样本（P = 0.11，N = 4和4个，分别）。因此，我们可以检查是否齿状回准确的解剖进行这种简单的实时PCR实验。 第三，以评估这一解剖方法的有效性，我们比较了整个海马齿状回的mRNA表达水平。全海马和齿状回野生的类型（n的= 9和4，分别）和ALPHA - CaMKII /获得 - 小鼠（n的= 18和4，分别）是芯片分析处理，并为所有基因的得分，在倍，变化是由野生型的值除以突变值计算。结果表明，mRNA表达的变化，特别是齿状回特定的分子，如DSP和TDO2，检测齿状回样品的灵敏度在整个海马样本（见表1）相比增加了5倍。我们以前表明，α- CaMKII + / -小鼠表现出的行为与人类精神失常，如工作记忆障碍和夸张infradian 节奏 1,5 。此外，突变小鼠齿状回神经元的形态和电生理功能不成熟齿状回神经元在正常老鼠惊人地相似，表明这些突变小鼠的神经元没有发展到成熟1。未成熟的齿状回和下调表达的DSP和TDO2 ALPHA - CaMKII表达+ / - 小鼠是符合的，DSP和TDO2可以被作为成熟颗粒细胞的标记使用的齿状回（大平等人发现，未发表。数据）。 两者合计，这个方便，准确的夹层技术可以可靠地用于研究侧重于齿状回。使用这种方法获得的齿状回组织是适用于其他类型的分析，包括蛋白质组学和细胞生物学分析。 <img src="/files/ftp_upload/1543/1543fig1.jpg" alt="图1" /> 图1。验证孤立的组织学研究的齿状回。尼氏染色（左图），和一个大脑后隔离齿状回的冠状断面处理示意图改编自小鼠大脑atlas6代表约在左侧面板中（右图）所示的部分相同的水平。箭头指示插入针尖的方向。比例尺，1毫米。 <img src="/files/ftp_upload/1543/1543fig2.jpg" alt="图2" /> 图2隔离齿状回实时PCR验证。齿状回和阿蒙小号喇叭从四个野生型小鼠的β-肌动蛋白，实时定量PCR TDO2，器Mrg1b和Tyro3处理。结果是± SEM手段。进行统计分析，采用S T检验，P值均遵循：β-肌动蛋白，P = 0.11; TDO2，P = 0.000023（** 1）; DSP，P = 0.0000030（** 2）; Mrg1b ，P = 0.000080 **（3）; Tyro3，P = 0.00017（** 4）。 表1。整个海马和齿状回的微阵列分析基因差异表达α- CaMKII整个海马齿状回和+ / -小鼠，通过计算在野生型小鼠中检测到的折叠变化来确定。统计学生S T测试的意义之间使用野生型和α- CaMKII + / -小鼠的数据进行了分析。其中的基因，其表达展出P &lt;0.05在α- CaMKII + / - 小鼠比野生型小鼠齿状回，前50个基因被列出。请注意，远远低于整个海马齿状回的样本数量。 AffyID，Affymetrix的探针标识符​​; CKII，α- CaMKII + / - 小鼠，野生型，野生型小鼠。 <table border="1"><tbody><tr><td></td><td></td><td></td><td></td><td colspan="2">齿状回（P &lt;0.05） WT：N = 4，CKII + / - ：N = 4 </td><td></td><td colspan="2">整个海马 WT：N = 9，CKII + / - ：N = 18 </td></tr><tr><td>基因标题</td><td>基因库</td><td> AffyID </td><td></td><td>折叠变化</td><td> P值</td><td></td><td>折叠变化</td><td> P值</td></tr><tr><td> desmoplakin </td><td> AV297961 </td><td> 1435494_s_at </td><td></td><td> 0.011018913 </td><td> 7.02694E - 06 </td><td></td><td> 0.037021003 </td><td> 1.86126E - 13 </td></tr><tr><td> desmoplakin </td><td> AV297961 </td><td> 1435493_at </td><td></td><td> 0.014369734 </td><td> 7.86747E - 06 </td><td></td><td> 0.04232106 </td><td> 1.00579E - 12 </td></tr><tr><td>色氨酸2,3 - 双加氧酶</td><td> AI098840 </td><td> 1419093_at </td><td></td><td> 0.020986484 </td><td> 5.23546E - 09 </td><td></td><td> 0.101037776 </td><td> 4.14823E - 13 </td></tr><tr><td> nephronectin </td><td> AA223007 </td><td> 1452106_at </td><td></td><td> 0.075479901 </td><td> 1.05191E - 08 </td><td></td><td> 0.234001154 </td><td> 1.66301E - 15 </td></tr><tr><td> nephronectin </td><td> AA223007 </td><td> 1452107_s_at </td><td></td><td> 0.079457767 </td><td> 1.40433E - 07 </td><td></td><td> 0.177974715 </td><td> 3.9758E - 12 </td></tr><tr><td>促甲状腺激素释放激素受体</td><td> M59811 </td><td> 1449571_at </td><td></td><td> 0.103105815 </td><td> 0.003093796 </td><td></td><td> 0.801412732 </td><td> 0.283994361 </td></tr><tr><td> 1，骨骼肌ryanodine受体</td><td> X83932 </td><td> 1427306_at </td><td></td><td> 0.104825517 </td><td> 3.38513E - 07 </td><td></td><td> 0.650685017 </td><td> 0.000308462 </td></tr><tr><td> nescient螺旋环螺旋1 </td><td> NM_010916 </td><td> 1419533_at </td><td></td><td> 9.431896 </td><td> 6.7979E - 06 </td><td></td><td> 4.078815314 </td><td> 5.27E - 11 </td></tr><tr><td> COPINE家庭成员九</td><td> BB274531 </td><td> 1454653_at </td><td></td><td> 9.159157 </td><td> 7.99492E - 06 </td><td></td><td> 1.797304153 </td><td> 0.000296375 </td></tr><tr><td> doublecortin - 3激酶</td><td> BB326709 </td><td> 1436532_at </td><td></td><td> 0.109336662 </td><td> 1.95278E - 07 </td><td></td><td> 0.56697229 </td><td> 2.62633E - 08 </td></tr><tr><td>蛋白酶3 </td><td> AF127766 </td><td> 1426043_a_at </td><td></td><td> 0.111269769 </td><td> 8.07053E - 06 </td><td></td><td> 0.370956608 </td><td> 2.04421E - 14 </td></tr><tr><td>成年男性纹状体的cDNA全长，日本理化学研究所丰富的图书馆，克隆：C030023B07产品：不能分类，插入序列</td><td> BB357628 </td><td> 1460043_at </td><td></td><td> 0.118712341 </td><td> 6.16926E - 07 </td><td></td><td> 0.682339204 </td><td> 2.33001E - 06 </td></tr><tr><td>胶原蛋白和钙离子结合表皮生长因子域1 </td><td> AV264768 </td><td> 1437385_at </td><td></td><td> 0.124043978 </td><td> 3.65669E - 05 </td><td></td><td> 0.488394112 </td><td> 4.05538E - 06 </td></tr><tr><td> β淀粉样蛋白（A4）的前体蛋白结合，一个家庭，成员2结合蛋白</td><td> AK013520 </td><td> 1431946_a_at </td><td></td><td> 7.7986307 </td><td> 1.2098E - 06 </td><td></td><td> 2.099164713 </td><td> 1.67047E - 06 </td></tr><tr><td> calbindin - 28K </td><td> BB177770 </td><td> 1456934_at </td><td></td><td> 0.130255444 </td><td> 3.32186E - 06 </td><td></td><td> 0.572605751 </td><td> 1.99157E - 10 </td></tr><tr><td>转录位点</td><td> AV328597 </td><td> 1443322_at </td><td></td><td> 0.133290835 </td><td> 5.43583E - 06 </td><td></td><td> 0.562767164 </td><td> 7.56544E - 06 </td></tr><tr><td>神经肽Y受体Y2 </td><td> NM_008731 </td><td> 1417489_at </td><td></td><td> 0.135319609 </td><td> 0.000113407 </td><td></td><td> 0.781498474 </td><td> 0.00394504 </td></tr><tr><td> ras基因反应元件结合蛋白1 </td><td> BE197381 </td><td> 1428657_at </td><td></td><td> 0.138235114 </td><td> 7.93691E - 07 </td><td></td><td> 0.651220705 </td><td> 2.94209E - 05 </td></tr><tr><td>胶质细胞源性神经营养因子家族受体的阿尔法2 </td><td> BB284482 </td><td> 1433716_x_at </td><td></td><td> 0.1390​​62563 </td><td> 2.35371E - 06 </td><td></td><td> 0.669544709 </td><td> 0.000214146 </td></tr><tr><td>脑啡肽1 </td><td> M13227 </td><td> 1427038_at </td><td></td><td> 6.9850435 </td><td> 2.39074E - 08 </td><td></td><td> 1.766018828 </td><td> 0.000250501 </td></tr><tr><td>日本理化学研究所基因1810010H24基因</td><td> BI729991 </td><td> 1428809_at </td><td></td><td> 6.8658915 </td><td> 1.88516E - 05 </td><td></td><td> 2.77573142 </td><td> 6.81865E - 09 </td></tr><tr><td> 1，骨骼肌ryanodine受体</td><td> BG793713 </td><td> 1457347_at </td><td></td><td> 0.151364292 </td><td> 3.35612E - 05 </td><td></td><td> 0.503144617 </td><td> 4.32907E - 05 </td></tr><tr><td> protocadherin 21 </td><td> NM_130878 </td><td> 1418304_at </td><td></td><td> 0.152671849 </td><td> 8.57783E - 06 </td><td></td><td> 0.670714726 </td><td> 1.56309E - 05 </td></tr><tr><td> cornichon同源3（果蝇） </td><td> NM_028408 </td><td> 1419517_at </td><td></td><td> 0.153724144 </td><td> 8.90755E - 06 </td><td></td><td> 0.95780695 </td><td> 0.661055608 </td></tr><tr><td> harakiri，BCL2基因相互作用蛋白（只包含BH3结构域） </td><td> BQ175572 </td><td> 1439854_at </td><td></td><td> 0.154284407 </td><td> 2.0118E - 05 </td><td></td><td> 0.56516812 </td><td> 4.86925E - 09 </td></tr><tr><td>碳水化合物（4-0）磺基转移酶的N -乙酰9 </td><td> AK017407 </td><td> 1431897_at </td><td></td><td> 0.155238951 </td><td> 5.37423E - 06 </td><td></td><td> 1.14910007 </td><td> 0.215637733 </td></tr><tr><td>蛋白酶3 </td><td> AI323605 </td><td> 1433681_x_at </td><td></td><td> 0.160871988 </td><td> 1.07655E - 05 </td><td></td><td> 0.477164757 </td><td> 1.33753E - 11 </td></tr><tr><td>锌指，远洋域含5 </td><td> BQ126004 </td><td> 1437355_at </td><td></td><td> 0.161812078 </td><td> 3.08262E - 06 </td><td></td><td> 0.421252632 </td><td> 0.01152969 </td></tr><tr><td> loricrin </td><td> NM_008508 </td><td> 1448745_s_at </td><td></td><td> 0.165129967 </td><td> 1.86362E - 05 </td><td></td><td> 0.639733409 </td><td> 0.000729772 </td></tr><tr><td> spondin 1，（F - spondin）细胞外基质蛋白</td><td> BC020531 </td><td> 1451342_at </td><td></td><td> 0.168035879 </td><td> 6.67867E - 07 </td><td></td><td> 0.821042412 </td><td> 0.023650765 </td></tr><tr><td>日本理化学研究所基因A930035E12基因</td><td> AV348640 </td><td> 1429906_at </td><td></td><td> 5.9086795 </td><td> 1.747E - 07 </td><td></td><td> 1.470383201 </td><td> 0.104085454 </td></tr></tbody><tbody><tr><td> BB247294日本理化学研究所全长丰富，7天的新生儿小脑小家鼠的cDNA克隆A730018G18 3&#39;，mRNA序列。 </td><td> BB247294 </td><td> 1447907_x_at </td><td></td><td> 5.9047494 </td><td> 1.04931E - 05 </td><td></td><td> 1.968147585 </td><td> 0.00010636 </td></tr><tr><td> FERM域包含3 </td><td> BB099015 </td><td> 1437075_at </td><td></td><td> 5.860216 </td><td> 0.000345581 </td><td></td><td> 2.780297178 </td><td> 1.83072E - 06 </td></tr><tr><td神经pentraxin 2 / / /假定蛋白LOC100044234 </td><td> NM_016789 </td><td> 1420720_at </td><td></td><td> 5.7568517 </td><td> 1.34227E - 06 </td><td></td><td> 2.652516957 </td><td> 0.000206279 </td></tr><tr><td>转录序列</td><td> BG076361 </td><td> 1460101_at </td><td></td><td> 5.657735 </td><td> 2.5015E - 06 </td><td></td><td> 1.296248831 </td><td> 0.22870031 </td></tr><tr><td> spondin 1，（F - spondin）细胞外基质蛋白</td><td> BC020531 </td><td> 1424415_s_at </td><td></td><td> 0.17783576 </td><td> 1.01658E - 06 </td><td></td><td> 0.836181248 </td><td> 0.001380141 </td></tr><tr><td> calbindin - 28K </td><td> BB246032 </td><td> 1448738_at </td><td></td><td> 0.180317904 </td><td> 1.35961E - 05 </td><td></td><td> 0.647334052 </td><td> 4.12268E - 09 </td></tr><tr><td> MARCKS，像1 </td><td> AV110584 </td><td> 1437226_x_at </td><td></td><td> 0.186235935 </td><td> 1.47067E - 06 </td><td></td><td> 0.499291387 </td><td> 2.34984E - 08 </td></tr><tr><td> matrilin 2 </td><td> BB338441 </td><td> 1455978_a_at </td><td></td><td> 0.187783528 </td><td> 6.19122E - 05 </td><td></td><td> 0.8967688 </td><td> 0.282337853 </td></tr><tr><td> matrilin 2 </td><td> BC005429 </td><td> 1419442_at </td><td></td><td> 0.188195795 </td><td> 0.000105295 </td><td></td><td> 0.915528892 </td><td> 0.35282097 </td></tr><tr><td> spondin 1，（F - spondin）细胞外基质蛋白</td><td> BQ175871 </td><td> 1442613_at </td><td></td><td> 0.189956563 </td><td> 9.41195E - 06 </td><td></td><td> 0.861033222 </td><td> 0.1394266 </td></tr><tr><td> 3休止，视网膜</td><td> NM_133205 </td><td> 1450329_a_at </td><td></td><td> 5.2130346 </td><td> 2.90599E - 05 </td><td></td><td> 3.944218329 </td><td> 1.07437E - 07 </td></tr><tr><td>日本理化学研究所基因A330050F15基因</td><td> AV325555 </td><td> 1457558_at </td><td></td><td> 0.19186781 </td><td> 0.000119342 </td><td></td><td> 0.660282035 </td><td> 2.47342E - 05 </td></tr><tr><td> contactin 3 </td><td> BB559510 </td><td> 1438628_x_at </td><td></td><td> 0.194404608 </td><td> 4.08641E - 07 </td><td></td><td> 0.918742591 </td><td> 0.022545297 </td></tr><tr><td> calbindin - 28K </td><td> BB246032 </td><td> 1417504_at </td><td></td><td> 0.196381321 </td><td> 2.24182E - 05 </td><td></td><td> 0.619305124 </td><td> 3.6222E - 06 </td></tr><tr><td>胃泌素释放肽</td><td> BC024515 </td><td> 1424525_at </td><td></td><td> 4.9436426 </td><td> 3.00588E - 05 </td><td></td><td> 2.752845903 </td><td> 5.72954E - 07 </td></tr><tr><td> sortilin相关VPS10域受体3 </td><td> AK018111 </td><td> 1425111_at </td><td></td><td> 4.885766 </td><td> 1.03645E - 05 </td><td></td><td> 1.29051599 </td><td> 0.029733649 </td></tr><tr><td>多巴胺受体D1A </td><td> BE957273 </td><td> 1455629_at </td><td></td><td> 4.869493 </td><td> 3.77525E - 05 </td><td></td><td> 1.815881979 </td><td> 0.000516498 </td></tr><tr><td> proprotein convertase枯草杆菌/可心类型5 </td><td> BB241731 </td><td> 1437339_s_at </td><td></td><td> 0.210528027 </td><td> 7.83039E - 05 </td><td></td><td> 0.574126078 </td><td> 9.15496E - 05 </td></tr></tbody><tbody><tr><td>白细胞介素1受体，I型</td><td> NM_008362 </td><td> 1448950_at </td><td></td><td> 0.210572243 </td><td> 9.64524E - 06 </td><td></td><td> 0.241135352 </td><td> 2.79816E - 08 </td></tr></tbody></table>

dissection of hippocampal dentate gyrus from adult mouse

海马是大脑中最广泛的研究领域，由于其重要的职能作用，在内存中处理和学习，其显着的神经元细胞的可塑性，并参与癫痫，神经退行性疾病和精神疾病之一。海马是不同的区域组成，其中主要包括颗粒神经元，阿蒙的号角，其中主要包括锥体神经元，和这两个地区的齿状回，解剖和功能的电路连接。许多不同的mRNA和蛋白质的选择性表达齿状回，齿状回的成年神经发生的部位，也就是新的神经细胞不断产生在成年齿状回。探讨具体齿状回的mRNA和蛋白表达，激光捕获显微切割经常被使用。这种方法有一定局限性，但是，如需要特殊仪器和复杂的处理程序。在这个录像协议中，我们展示了一个从成年鼠的体视显微镜下齿状回的夹层技术。齿状回，准备使用这种技术的样本，适用于任何实验，包括转录组，蛋白质组和细胞生物学分析。我们确认，解剖组织进行实时PCR齿状回的特定基因，色氨酸2,3 - 双加氧酶（TDO2）和desmoplakin（DSP），和亚扪人的号角丰富的基因，MEIS相关基因1B（Mrg1b齿状回）和TYRO3酪氨酸蛋白激酶3（Tyro3）。 TDO2和DSP在齿状回样品的mRNA的表达明显较高的水平，而Mrg1b和Tyro3水平较低，比那些在阿蒙的号角样本，检测。为了证明这种方法的优点，我们进行DNA芯片分析整个海马及齿状回的样品。 TDO2和DSP的mRNA的表达，是在齿状回表达选择性在整个海马的α- CaMKII / - 小鼠，展出分别为0.037和野生型小鼠相比，0.10倍的变化。然而，在孤立的齿状回，这些表达式展出0.011和0.021倍的变化，野生型小鼠相比，表明在齿状回的基因表达的变化，可以用更高的灵敏度检测。两者合计，这个方便，准确的夹层技术可以可靠地用于研究侧重于齿状回。

Watch this Scientific Journal Video about Dissection of Hippocampal Dentate Gyrus from Adult Mouse at JoVE.com

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

一个去除从成年小鼠齿状回立体显微镜下解剖技术被证明在这个录像的协议。

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

dissection-of-hippocampal-dentate-gyrus-from-adult-mouse

Research

JoVE Journal

Biology

82.2K 次观看.  Japan Science and Technology Agency, Core Research for Evolutionary Science and Technology (CREST). 一个去除从成年小鼠齿状回立体显微镜下解剖技术被证明在这个录像的协议。

视频: Dissection of Hippocampal Dentate Gyrus from Adult Mouse

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

6.5 DPC小鼠胚胎剖析

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

科研

生物学

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

剖析器官的成年斑马鱼

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

从成人Oenocytes剖析果蝇</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

成年斑马鱼肾脏的解剖

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

来自新生儿和成年鼠皮肤的原代小鼠角化细胞的分离和培养

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

从成人小鼠腭中分离和培养原代口腔角质形成细胞

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.