A major problem in neurobiology concerns the mechanisms by which damaged neurons may re-grow axons and form selective connections to restore function. We propose to test the role of migrating microglia in successful axonal regeneration that follows injury to the leech nervous system. Migration of microglia occurs promptly, accounts for the increase in cell numbers at the lesion, and appears influenced by nitric oxide synthase activity, which is rapidly up regulated at the lesion. Microglial migration is also affected by applied electric fields of a size consistent with injury currents we measure at the lesion. Evidently microglia deposit laminin, a component of the extracellular matrix that promotes axon growth. The leech is particularly advantageous for these studies because (1) its ganglia contain identifiable neurons capable of regenerating specific connections following axotomy, (2) individual microglia can be tracked and their movements charted minute by minute, and (3) adult and embryonic nervous systems can be manipulated and examined both in vivo and in vitro. Experiments that interfere with and block accumulation of microglia at injury sites will test the role of microglia in sprouting and regeneration. Immunocytochemistry has shown abundant laminin transiently in the embryonic leech nervous system along axon pathways. Following injury to the adult nervous system, laminin reappears (first in patches at the lesion, later in streaks) in advance of axons. Whether the microglia that migrate toward the site of the lesion produce the laminin, as in vitro, will be determined using antibodies and in situ hybridization with our riboprobes for laminin. We will determine whether the streaks of laminin, which may guide regenerating axons, mark the paths of migrating microglial cells; fluorescently labeled microglia will be tracked in living preparations using low-light video microscopy. Alterations of nitric oxide levels and its synthesis and of electric fields such as those generated by the lesion will determine the actions of nitric oxide and fields in regulating microglial migration. These studies will clarify the roles of microglia and of laminin in axonal regeneration following injury, and may suggest strategies for achieving equally successful axonal repair in the mammalian nervous system.
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