Hagihara Hideo, Toyama Keiko, Yamasaki Nobuyuki, Miyakawa Tsuyoshi
Division of Systems Medical Science, Institute for Comprehensive Medical Science, Fujita Health University.
J Vis Exp. 2009 Nov 17(33):1543. doi: 10.3791/1543.
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.
海马体是大脑中研究最为广泛的区域之一，这是因为它在记忆处理和学习中发挥着重要的功能作用，具有显著的神经元细胞可塑性，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构成，海马角主要由锥体神经元构成，这两个区域通过解剖学和功能回路相连。许多不同的mRNA和蛋白质在齿状回中选择性表达，并且齿状回是成体神经发生的部位；也就是说，成体齿状回中不断有新的神经元生成。为了研究齿状回特有的mRNA和蛋白质表达，常使用激光捕获显微切割技术。然而，这种方法有一些局限性，比如需要特殊设备和复杂的操作程序。在这个视频记录的方案中，我们展示了一种在体视显微镜下从成年小鼠中分离齿状回的解剖技术。使用这种技术制备的齿状回样本适用于任何检测，包括转录组学、蛋白质组学和细胞生物学分析。我们通过对齿状回特异性基因色氨酸2,3-双加氧酶（TDO2）和桥粒斑蛋白（Dsp）以及海马角富集基因Meis相关基因1b（Mrg1b）和酪氨酸蛋白激酶3（Tyro3）进行实时PCR，证实解剖得到的组织是齿状回。与海马角样本相比，齿状回样本中TDO2和Dsp的mRNA表达水平明显更高，而Mrg1b和Tyro3的表达水平更低。为了证明这种方法的优势，我们使用整个海马体和齿状回的样本进行了DNA微阵列分析。在α-CaMKII+/-小鼠的整个海马体中，选择性表达于齿状回的TDO2和Dsp的mRNA表达与野生型小鼠相比，分别呈现0.037和0.10倍的变化。然而，在分离的齿状回中，这些表达与野生型小鼠相比呈现0.011和0.021倍的变化，这表明可以更灵敏地检测到齿状回中的基因表达变化。综上所述，这种方便且准确的解剖技术可可靠地用于专注于齿状回的研究。
