The experiments of this proposal are designed to identify the proteins produced within the neuropil of the dentate gyrus during periods of neuronal sprouting and synapse growth. The long term objective of this approach is to define some of the molecular processes that may be involved in synaptogenesis during normal development and in response to lesion. Proposed experiments are based on four discoveries about the model system (rat's dentate gyrus): 1) 3H-leucine incorporation into protein increases over the denervated dentate neuropil concurrent with synapse formation, 2) most of the polyribosomes in the dentate neuropil are associated with dendritic spines, 3) the number of spine associated polyribosomes is high during developmental synaptogenesis, decreasing as the synapse matures and 4) the incidence of spine associated polyribosomes increases during reactive synaptogenesis. The working hypotheses are: A) local synthesis of proteins within the neuropil plays a key role in regulation of synapse growth, and B) part of the local synthesis is carried out by the polyribosomes that are selectively positioned beneath synapses. We will begin to address these hypotheses using in situ hybridization to determine the distribution of selected mRNAs within the dentate gyrus during synapse formation and reinnervation. As a second index of local synthesis we will determine tissue levels of the same proteins within dissected neuropil and cell body layers. Finally, using the hippocampal slice preparation we will radiolabel polypeptides made in the dentate neuropil and then apply standard biochemical methods (i.e., 1 and 2 dimensional gel electrophoresis and immunoblotting) to characterize the newly synthesized proteins. This approach will tell us: 1) whether mRNAs for major cytoskeletal and synaptic proteins vary as a function of reactive synaptogenesis, 2) if these are regional differences in the level of these proteins within the granule neurons and 3) what profile of proteins are made locally within the dentate neuropil and how these compare to those synthesized in the cell body.
| Phillips, L L; Belardo, E T (1994) Increase of c-fos and ras oncoproteins in the denervated neuropil of the rat dentate gyrus. Neuroscience 58:503-14 |
| Phillips, L L; Belardo, E T (1992) Expression of c-fos in the hippocampus following mild and moderate fluid percussion brain injury. J Neurotrauma 9:323-33 |
| Phillips, L L; Pollack, A E; Steward, O (1990) Protein synthesis in the neuropil of the rat dentate gyrus during synapse development. J Neurosci Res 26:474-82 |
| Phillips, L L; Steward, O (1990) Increases in mRNA for cytoskeletal proteins in the denervated neuropil of the dentate gyrus: an in situ hybridization study using riboprobes for beta-actin and beta-tubulin. Brain Res Mol Brain Res 8:249-57 |
