The Process of Reinnervation in the Dentate Gyrus of Adult Rats: Temporal Relationship between Changes in the Levels of Glial Fibrillary Acidic Protein (GFAP) and GFAP mRNA in Reactive Astrocytes

Abstract

The present study evaluates the temporal relationships between increases in glial fibrillary acidic protein (GFAP) mRNA, GFA protein levels, and GFAP immunostaining in the hippocampus of adult rats following unilateral lesions of the entorhinal cortex (EC). GFAP mRNA levels were assessed at 12 h, 1, 2, 4, 6, 8, 10, 12, 14, and 30 days postlesion by dot blot assays using 35 S-labeled cRNA probes against the mRNA. Animals were also prepared for in situ hybridization during the peak of GFAP mRNA expression (2 days postlesion) to explore the nature of individual differences in the spatial extent of the increases. GFA protein levels were assessed by Western blot and dot immunoblot techniques in a separate group of animals prepared at 1, 2, 4, 6, 8, and 10 days postlesion and by immunostaining at 1, 2, 4, 6, and 8 days postlesion. The dot blot analyses of GFAP mRNA levels confirmed previous studies, in that we observed dramatic increases in the levels of GFAP mRNA in the hippocampus ipsilateral to the EC lesions. The increases were biphasic, with a large peak in mRNA levels at 1-2 days postlesion (about 10-fold greater than control) and a second peak at 6-8 days. In most animals, the increases were predominantly ipsilateral to the lesion. However, in some animals, there were also large increases on the contralateral side. In situ hybridization experiments revealed two different spatial patterns of increased gene expression, one in which the increases in GFAP mRNA occurred bilaterally and one in which increases were restricted primarily to the hippocampus ipsilateral to the lesion. Immunochemical measures revealed that GFA protein levels increased gradually in the hippocampus ipsilateral to the lesion, reaching a peak at about 2-fold higher than control at 4 days postlesion, and then remained near this level until at least 10 days postlesion. In the contralateral hippocampus, GFA protein levels were increased to about the same extent as on the ipsilateral side at 1, 2, and 4 days postlesion, but then began to decline, returning to near control levels by 8 days. Increases in immunostaining occurred with about the same time course as the increases in GFA protein levels as measured immunochemically. These results define the temporal relationship between increases in GFAP mRNA and increases in GFA protein, providing new insights into the regulation of gene expression in reactive astrocytes.