The goal of this proposal (ECM) molecules that are potent promoters of neurite outgrowth in monolayer cultures of dissociated CNS neurons. However, it has been difficult to examine whether laminins promote axon outgrowth in vivo, because the elimination of laminins expression in transgenic (knockout) animals results in an embryonic lethal phenotype. In order to examine the function of laminins in well-organized brain tissue, we have designed experiments using cultured hippocampal slices, whose anatomy and circuitry are remarkably similar to the intact hippocampus. Hippocampal slice cultures, therefore, provide a powerful model system for analyzing axon regeneration of CNS neurons under well-controlled in vitro conditions. We have chosen to study the MF pathway because it possesses profound plasticity and regenerative properties in vivo and in vitro. Indeed, MFs are capable of regenerating their normal synaptic pattern following transection in slice cultures, and they retain a plastic form of sprouting when transected in vivo. Significantly, our preliminary data suggest the laminin is expressed along the MF pathway and termination zone. The regenerative abilities of MFs make this pathway an ideal choice for examining the growth and guidance promoting effects of laminins on CNS axons. Our preliminary data demonstrate that application of antisense oligodeoxyribonuclotides (ODNs), which inhibits laminin expression, produces severe retardation and misguidance of transected MFs in slice cultures made from P4 animals. We now propose to perform a detained analysis of this abnormal regeneration using image analysis and confocal microscopy of stained MFs and terminals in fixed sections. MFs in living tissue will be analyzed using time-lapse imaging. The maximum period of MF outgrowth is between P0 and P14, and the pathway exhibits mature morphology by P21. Slices made from P21 animals will be examined to determine if MF regeneration is limited to a """"""""critical period"""""""" that corresponds to the time of maximal MF outgrowth (P0-P14). In order to identify the specific subtypes of laminins and their integrin receptors that control axon regeneration, a detailed analysis of laminins and integrin subunit expression will be performed. We will also examine the role of laminins in directing MF outgrowth from hippocampal progenitor cells transplanted onto slice cultures. These progenitor cells have been shown to differentiate into dentate granule neurons when transplanted into the hippocampus of intact animals. Understanding the molecules that enable the mossy fiber pathway to regenerate may allow us to develop better transplantation and bridge building strategies that can be used in CNS pathways that fail to regenerate.
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