Increase of c-fos and ras oncoproteins in the denervated neuropil of the rat dentate gyrus

Abstract

When the entorhinal cortical input to the rat dentate gyrus is destroyed, the process of sprouting and synaptogenesis begins within the denervated dendritic laminae. The present study used immuno-histochemical methods to determine whether there was an increase in the oncoproteins c-fos and ras within the denervated neuropil of the dentate gyrus during this period of terminal growth and synapse formation. Animals were prepared for immunolabeling one, three, six and 30 days after unilateral lesion of the entorhinal cortex. Rats were perfused with paraformaldehyde fixative and brain sections were incubated with antibodies to either c-fos or ras oncoprotein. Qualitative light microscopic analysis showed a marked increase in both c-fos and ras proteins over the denervated zone at three days postlesion when compared to both the intact contralateral control and the naive control. At one- and six-day postlesion intervals there was also an increase in labeling over the denervated neuropil with each oncoprotein; however, the intensity of label was reduced relative to that of the three-day time interval. No increase in labeling over the denervated zone was visible for either antibody at 30 days postlesion. The high level of both c-fos and ras labeling in the denervated molecular layer was confirmed with Western blot analysis of dissected molecular layers from lesioned and contralateral control hippocampi. Controls for antibody and method specificity showed that the labeling was specific for c-fos and ras proteins. The high level of c-fos labeling over the denervated molecular layer was uniform, with scattered punctate sites of reaction product interspersed in the neuropil. Glial cell bodies in the neuropil contained the highest levels of c-fos oncoprotein. The granule cell nuclei showed an apparent reduction in the level of c-fos labeling at one, three and six days postlesion when compared with the nuclear staining of naive control cases. At 30 days postlesion, high levels of labeling over the denervated zone were not visible and c-fos localization had returned to the typical predominant nuclear sites seen in controls. Ras oncoprotein localization was diffuse in the cell processes of the molecular layer, with intermittent glial labeling within the denervated zone. No cell nuclei labeling was observed with antibodies to ras protein. These results show that both c-fos and ras oncoproteins are increased within the denervated neuropil of the dentate gyrus during sprouting and synapse formation. Our study suggests that neuropil alterations in gene expression through c-fos, and membrane signal transduction by ras may be part of the cellular mechanisms invoked during lesion-induced synaptogenesis.