Casoli Tiziana, Stefano Giuseppina Di, Fattoretti Patrizia, Solazzi Moreno, Delfino Alessia, Biagini Graziella, Bertoni-Freddari Carlo
Neurobiology of Aging Center, N. Masera INRCA Research Department, Via Birarelli 8, Ancona 60121, Italy.
Micron. 2003;34(8):415-22. doi: 10.1016/S0968-4328(03)00038-6.
The growth-associated protein GAP-43 is a presynaptic membrane phosphoprotein that is expressed at high levels during development and axonal growth. To evaluate the cellular distribution of GAP-43 mRNA in the hippocampus and cerebellum of adult rats we applied in situ hybridization (ISH) as well as direct and indirect in situ RT-PCR using biotin as a reporter molecule. ISH resulted in a positive signal in most cerebellar granular cells and in 30% of hippocampal CA3 neurons. Direct in situ RT-PCR yielded cells with strong signals in every region investigated, with elevated background levels most likely related to incorporation of labeled nucleotides into non-specific amplicons through internal priming and DNA repair activity. Indirect in situ RT-PCR turned out to be the best approach for detecting GAP-43 mRNA positive cells. Cerebellar cells exhibiting a positive signal for GAP-43 mRNA were of the granular cell type (98%). Hippocampal neurons with a positive reaction for GAP-43 mRNA included all the neuron groups analyzed, namely CA1 (99%) and CA3 pyramidal cells (94%) and dentate gyrus granule cells (92%). Dentate gyrus granule cells have not tested positive for GAP-43 mRNA detection by molecular morphology analysis. These data show that in normal rats GAP-43 mRNA is present in different cell populations of hippocampal formation, supporting the role of this protein in the ongoing processes of synaptic plasticity.
生长相关蛋白GAP - 43是一种突触前膜磷蛋白，在发育和轴突生长过程中高水平表达。为了评估成年大鼠海马体和小脑中GAP - 43 mRNA的细胞分布，我们应用了原位杂交（ISH）以及使用生物素作为报告分子的直接和间接原位逆转录聚合酶链反应（RT - PCR）。ISH在大多数小脑颗粒细胞和30%的海马体CA3神经元中产生阳性信号。直接原位RT - PCR在每个研究区域都产生了强信号的细胞，背景水平升高很可能与通过内部引物和DNA修复活性将标记核苷酸掺入非特异性扩增子有关。间接原位RT - PCR被证明是检测GAP - 43 mRNA阳性细胞的最佳方法。显示GAP - 43 mRNA阳性信号的小脑细胞为颗粒细胞类型（98%）。对GAP - 43 mRNA呈阳性反应的海马神经元包括所有分析的神经元群体，即CA1（99%）和CA3锥体细胞（94%）以及齿状回颗粒细胞（92%）。通过分子形态学分析，齿状回颗粒细胞在GAP - 43 mRNA检测中未呈阳性。这些数据表明，在正常大鼠中，GAP - 43 mRNA存在于海马结构的不同细胞群体中，支持了该蛋白在持续的突触可塑性过程中的作用。
