Developmental changes of RNA editing of glutamate receptor subunits GluR5 and GluR6: in vivo versus in vitro

Abstract

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 developmental 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.