Mammalian dorsal root axons regenerate robustly within the PNS environment of the root, yet the majority are stopped at the root- cord interface. It has long been maintained that reactive astrocytes in this region prohibit the elongation of axons into the cord. Although a number of hypotheses have been proposed to explain the astrocytic role in this inhibition of axonal elongation, none of these hypotheses have been rigorously tested. I propose to test the novel hypothesis that reactive astrocytes prevent axonal elongation through the region of the root entry zone by activating physiological mechanisms within the axon tips, much as normal targets activate these mechanisms. This hypothesis is based on the idea that there is a physiological interaction between growth cones and adult mammalian astrocytes and, contrasts with the long held idea that astrocytes in the root entry zone merely act as an inert physical barrier. To discriminate between physiologically activated axon terminals and physically blocked terminals, I will use the accumulation of neurofilaments as an assay. The basis for this assay is the fact that normal axon terminals are devoid of neurofilaments because the neurofilaments are continuously degraded by endogenous proteases that are activated by interactions with target cells. By contrast, when growing axons are blocked by a physical barrier, as in a ligation neuroma, where targets are unavailable, neurofilaments that are continuously transported down the axon, accumulate and swell the trapped axon terminals. My preliminary studies of regenerating dorsal root axons in adult rats have shown that axons that stop at the root-cord interface form terminals upon astrocytes that persist for months in a non- growing steady state. Like normal synapatic terminals these axo- glial terminals are characterized by the absence of neurofilaments. In the proposed study, I will use morphological and biochemical techniques, in conjunction with protease inhibitors, to analyze these steady state axon terminals and their interaction with the astrocytes at the root-cord interface. Moreover, I will compare and contrast the terminals of the stopped axons at the root-cord interface with those at the physical barrier of the ligation neuroma. The result of these analyses will reveal basic processes by which astrocytes regulate axonal regeneration in the adult mammalian CNS.
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