Protein synthesis in the neuropil of the rat dentate gyrus during synapse development

Abstract Previous studies have shown that there are dramatic accumulations of polyribosomes under developing synapses on dendrites of CNS neurons. The present study was designed to evaluate what types of proteins might be synthesized by the synapse‐associated polyribosomes. Hippocampal slics from rat pups sacrificed at 4,7,10,12,14, and 21 days after birth as well as slices from adult animals were incubated in a modified Eagle's medium containing 3 H‐leucine. After a 30 min exposure to radiolabeled amino acids, the slices were microdissected, separating the dendritic enriched molecular layer from the cell bodies of the dentate gyrus and the hippocampus proper. The level of protein synthetic activity was assessed by comparing the incorporation in cell body and dendritic laminae. Polypeptides present in each dissected zone were separated electrophoretically on 1D SDS‐polyacrylamide gels according to their molecular weight and the newly synthesized proteins were analyzed through gel fluorography. The overall level of 3 H‐amino acid incorporation into protein (measured as cpm/μg protin) was higher than that of the adult at all postnatal ages. When the entire slice was analyzed, the maximum incorporation was at 12 days after birth. In the dissected subregions of the slice the peak protein synthetic activity in cell‐body‐enriched regions of dentate gyrus and hippocampus proper was at 4–7 days postnatal, declining between 7 and 21 days to values comparable to the adult. By contrast, protein synthesis in the molecular layer of the dentate gyrus did not peak until 12 days after birth, decreasing toward adult rates after 14 days. The overall pattern of Coomassie stained polypeptides present in the dentate molecular layer was comparable at all ages examined. Moreover, one‐dimensional gel analysis showed no qualitative differences in the proteins that were synthesized in the three dissected zones across ages. Gel fluorography identified four mid‐molecular weight ranges of polypeptides (43, 50, 52, and 56 kD) and a group of high molecular weight proteins (190‐220 kD) that were the most heavily labeled species in the dissected molecular layer. Quantitative gel analysis revealed that three of the midrange bands (43, 52, and 56 kD) accounted for a different percentage of the over all labeled protein across developmental age; prominent changes were found in the 43 kD band, where increased radiobeling was directly correlated with postnatal periods of rapid synaptogenesis. We conclude that there is a high level of protein synthesis in dendritic laminae during developmental synaptogenesis. This burst of synthesis appears to result in the increased generation of a set of proteins which are normally synthesized within both granule cell body and dendrite enriched regions. The 43 kD member (s) of this set of polypeptides shows increased labeling during the most rapid phase of synaptogenesis.