Dissection of hippocampal dentate gyrus from adult mouse.
Dissection of hippocampal dentate gyrus from adult mouse.
复制标题
DOI：
10.3791/1543
复制
发表时间：
2009-11-17
期刊：
影响因子：
--
通讯作者：
Miyakawa, Tsuyoshi
中科院分区：
文献类型：
--
作者：
Hagihara, Hideo;Toyama, Keiko;Miyakawa, Tsuyoshi
海马区是脑内研究最广泛的区域之一，因为它在记忆处理和学习中具有重要的功能作用，它具有显著的神经细胞可塑性，并且与癫痫、神经退行性疾病和精神障碍有关。海马体由不同的区域组成；齿状回，主要由颗粒神经元组成；阿蒙角，主要由锥体神经元组成，这两个区域通过解剖和功能回路连接。许多不同的mRNAs和蛋白质选择性地表达在齿状回中，而齿状回是成体神经发生的场所，也就是说，成体齿状回中不断地产生新的神经元。为了研究齿状回特异的mRNA和蛋白表达，经常使用激光捕获显微解剖。然而，这种方法有一些局限性，如需要特殊的仪器和复杂的处理程序。在这个录像方案中，我们演示了一种在立体显微镜下从成年小鼠身上移除齿状回的解剖技术。用这种技术制备的齿状回样本适用于任何分析，包括转录组、蛋白质组和细胞生物学分析。我们通过对齿状回特异基因色氨酸2，3-双加氧酶(TDO2)和桥粒蛋白(DSP)以及Ammon的角蛋白丰富基因Meis相关基因1b(MRG1b)和Tyro3蛋白酪氨酸激酶3(Tyro3)的实时荧光聚合酶链式反应(Real-time PCR)确认所解剖的组织是齿状回。海马齿状回标本中TDO2和DSPmRNA的表达水平明显高于海马齿状回标本，而MRG1b和Tyro3的表达水平则明显低于海马齿状回标本。为了证明这种方法的优势，我们使用整个海马区和齿状回的样本进行了DNA微阵列分析。α-CaMKII+/-小鼠海马齿状回选择性表达的TDO2和DSPmRNA表达分别是野生型小鼠的0.037倍和0.1倍。然而，在分离的齿状回中，这些表达分别是野生型小鼠的0.011倍和0.021倍，这表明可以更敏感地检测到齿状回中基因表达的变化。综上所述，这种方便而准确的解剖技术可以可靠地用于对齿状回的研究。
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 remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.