Abstract

Axons in the adult mammalian CNS normally do not regenerate following axotomy even though they retain the capacity for growth under certain experimental conditions. Although this implies that the regeneration of adult axons is under regulative control, very little is known about the signaling pathways responsible for this regulation. This study examines the possibility that a G protein signaling system exists in adult mouse optic fibers and that it functions to regulate axonal outgrowth. To induce the growth of optic fibers, retinas from adult mouse were placed in organotypic culture under serum free conditions and allowed to regenerate onto a laminin substrate. Heterotrimeric G proteins were stimulated by adding mastoparan (MST) to the medium while monitoring growing fibers with time lapse microscopy. Mastoparan treatment produced rapid growth cone collapse and axonal retraction which persisted while MST was present. Prior addition of pertussis toxin (PTX), which irreversibly inactivates the G proteins, G o and G i, completely blocked the effect of MST, confirming that MST was acting through the PTX sensitive G proteins. Selective activation of G proteins in the growth cone by local application of MST with a micropipet was equally effective. For comparison, equivalent experiments were performed on embryonic day 15 retinal explants and on retinal explants from adult goldfish, which normally regenerate in vivo. MST similarly inhibited these axons and this effect was blocked by PTX. However, embryonic fibers were less reliably affected compared to goldfish or adult mouse, suggesting a developmentally regulated sensitivity. The presence of G-proteins in the mouse axons was further tested immunohistochemically using antibodies against. G o/G i. Positive staining was detected in the growth cones and shaft of adult and embryonic mouse optic fibers. These findings demonstrate that G protein activation inhibits axonal outgrowth and suggest that there may be a G protein signaling pathway that normally regulates this outgrowth. However, since this pathway appears to exist in both axons that can regenerate and those that normally do not, the presence of PTX-sensitive G proteins alone cannot account for regenerative failure. Regenerative failure may instead be explained as the selective or increased activation of this pathway in the adult mammalian CNS.

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