Paschen W, Schmitt J, Gissel C, Dux E
Max-Planck-Institute for Neurological Research, Department of Experimental Neurology, Köln, Germany.
Brain Res Dev Brain Res. 1997 Feb 20;98(2):271-80. doi: 10.1016/s0165-3806(96)00193-9.
In the present series of experiments we compared the up-regulation of GluR5 and GluR6 mRNA editing during the transition from the embryonic to the adult state with changes in the extent of editing during neuronal development in vitro. RNA was isolated from rats, from the cerebral cortex, hippocampus and cerebellum of embryonic brains (E19) and adult brains (2 months old), as well as from neurons prepared from the cortex, hippocampus and cerebellum of embryonic brains (E19) and held in tissue culture for 2, 8 or 16 days. Quantification of mRNA editing was achieved by using standards prepared from plasmids with cDNA inserts derived from the edited and unedited state of both GluR5 and GluR6 mRNA. In addition, GluR5 mRNA levels were determined in brain tissue and neuronal cells in culture by quantitative PCR. Developmental changes in the extent of GluR5 and GluR6 mRNA editing were different in vivo compared to in vitro. For GluR5 mRNA editing these differences were most pronounced in cerebellar neurons compared to cerebellar tissue: the extent of GluR5 mRNA editing found in vivo at E19 was significantly down-regulated in cerebellar neurons during the first 8 days in culture, and after 16 days in vitro the extent of editing was still about 50% of that found in the adult state in vivo. For GluR6 mRNA editing these differences were most pronounced in hippocampal neurons compared to the hippocampus in vivo: the extent of GluR6 mRNA editing found in vivo at E19 was significantly down-regulated in vitro during the whole culturing period, most pronounced after 8 days in vivo (to below 40% of that found at E19 and to below 30% of that found in adult hippocampus). GluR5 mRNA levels increased markedly from E19 to the adult brain. However, we could not find any specific pattern of changes in mRNA levels which might account for the development changes in the profile of GluR5 mRNA editing. Comparing developmental changes in the extent of mRNA editing of glutamate receptor subunits may help to elucidate the molecular and regulatory mechanisms of this important editing reaction. Strict control and clear indication of the age of primary neuronal cell cultures used should be required in accounts of electrophysiological or neurotoxicological studies as this would increase comparative usefulness of such experiments, since calcium fluxes through glutamate receptor ion channels are likely to influence the system significantly.
在本系列实验中，我们比较了从胚胎状态到成年状态转变过程中GluR5和GluR6 mRNA编辑的上调情况，以及体外神经元发育过程中编辑程度的变化。从大鼠胚胎脑（E19）和成年脑（2个月大）的大脑皮质、海马体和小脑中分离RNA，以及从胚胎脑（E19）的皮质、海马体和小脑中制备并在组织培养中培养2、8或16天的神经元中分离RNA。通过使用从含有GluR5和GluR6 mRNA编辑和未编辑状态的cDNA插入片段的质粒制备的标准品来实现mRNA编辑的定量。此外，通过定量PCR测定脑组织和培养的神经元细胞中GluR5 mRNA的水平。与体外相比，体内GluR5和GluR6 mRNA编辑程度的发育变化有所不同。对于GluR5 mRNA编辑，与小脑组织相比，这些差异在小脑神经元中最为明显：在培养的前8天，小脑神经元中E19时体内发现的GluR5 mRNA编辑程度显著下调，体外培养16天后，编辑程度仍约为成年状态下体内发现的编辑程度的50%。对于GluR6 mRNA编辑，与体内海马体相比，这些差异在海马神经元中最为明显：E19时体内发现的GluR6 mRNA编辑程度在整个培养期间在体外显著下调，在培养8天后最为明显（降至E19时发现的编辑程度的40%以下，以及成年海马体中发现的编辑程度的30%以下）。从E19到成年脑，GluR5 mRNA水平显著增加。然而，我们没有发现任何可能解释GluR5 mRNA编辑谱发育变化的mRNA水平变化的特定模式。比较谷氨酸受体亚基mRNA编辑程度的发育变化可能有助于阐明这种重要编辑反应的分子和调节机制。在电生理或神经毒理学研究报告中，应严格控制并明确指出所用原代神经元细胞培养物的年龄，因为这将增加此类实验的比较有用性，因为通过谷氨酸受体离子通道的钙通量可能会对该系统产生显著影响。
