INCREASE OF C-FOS AND RAS ONCOPROTEINS IN THE DENERVATED NEUROPIL OF THE RAT DENTATE GYRUS

When the entorhinal cortical input to the rat dentate gyrus is destroy ed, the process of sprouting and synaptogenesis begins within the dene rvated dendritic laminae. The present study used immunohistochemical m ethods to determine whether there was an increase in the oncoproteins c-fos and ras within the denervated neuropil of the dentate gyrus duri ng this period of terminal growth and synapse formation. Animals were prepared for immunolabeling one, three, six and 30 days after unilater al lesion of the entorhinal cortex. Rats were perfused with paraformal dehyde fixative and brain sections were incubated with antibodies to e ither c-fos or ras oncoprotein. Qualitative light microscopic analysis showed a marked increase in both c-fos and ras proteins over the dene rvated zone at three days postlesion when compared to both the intact contralateral control and the naive control. At one- and six-day postl esion intervals there was also an increase in labeling over the denerv ated neuropil with each oncoprotein; however, the intensity of label w as reduced relative to that of the three-day time interval. No increas e 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 anal ysis of dissected molecular layers from lesioned and contralateral con trol hippocampi. Controls for antibody and method specificity showed t hat the labeling was specific for c-fos and ras proteins. The high lev el 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 leve ls of c-fos oncoprotein. The granule cell nuclei showed an apparent re duction in the level of c-fos labeling at one, three and six days post lesion when compared with the nuclear staining of naive control cases. At 30 days postlesion, high levels of labeling over the denervated zo ne were not visible and c-fos localization had returned to the typical predominant nuclear sites seen in controls. Ras oncoprotein localizat ion was diffuse in the cell processes of the molecular layer, with int ermittent glial labeling within the denervated zone. No cell nuclei la beling was observed with antibodies to ras protein. These results show that both c-fos and ras oncoproteins are increased within the denerva ted neuropil of the dentate gyrus during sprouting and synapse formati on. Our study suggests that neuropil alterations in gene expression th rough c-fos, and membrane signal transduction by ras may be part of th e cellular mechanisms invoked during lesion-induced synaptogenesis.