PMC:529315 / 13614-16450 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/529315","sourcedb":"PMC","sourceid":"529315","source_url":"http://www.ncbi.nlm.nih.gov/pmc/529315","text":"To test the hypothesis that NT-3 acts as a chemoattractant for sensory axons, we performed a series of in vitro assays. Proprioceptive axons in mice enter the gray matter in the spinal cord and advance ventrally parallel to the midline by E13 and reach the motor neurons by E15 (Ozaki and Snider 1994). We co-cultured collagen-embedded E13 WT DRG explants with NT-3-soaked sepharose beads (n = 26). Control cultures were set up using bovine serum albumin (BSA)– or phosphate buffer saline (PBS)–soaked beads (n = 12). DRG axons began extending towards the localized NT-3 source by the end of the first day and consistently displayed a strong chemoattraction by 3 d in vitro, whereas they did not show such preference for BSA-loaded control beads (Figure 5A and 5B). This attraction was not due to survival support of NT-3 because Bax null ganglia displayed the same chemoattraction (Figure 5C; n = 16). NT-3 may act through either TrkC, or the p75NTR. We repeated the co-culture experiments with DRG explants derived from p75NTR KO mice (n = 18). Axons of these ganglia also showed strong chemoattraction towards the NT-3 beads (Figure 5D). Finally, we used diffusible TrkC receptors conjugated to IgG constant regions (TrkC-Fc) added to the medium (n = 6) to deplete soluble NT-3 from the collagen gels (Figure 5E). In the presence of TrkC-Fc the chemoattraction was completely blocked, demonstrating that the effect we see is specific for NT-3. In order to investigate the active range of our beads, we have repeated the cultures with WT E13 DRGs by placing the beads at increasing distances from the ganglia (n = 4 each) (Figure 5F). There was still preferred growth towards the bead at 1,200μm, the longest distance studied, although the number of axons and the extent of growth were not as robust. Next, we set up E13 DRG spinal cord explant cultures using NT-3-loaded beads placed at the midline at mid-spinal cord level as an ectopic NT-3 source (n = 15). Control cultures were set using PBS-loaded beads (n = 6). DiI labeling through the DRGs revealed numerous fibers entering the spinal cord at ectopic regions and growing towards the NT-3 beads (Figure 6A), surrounding the beads and forming bundles around them (Figure 6C–6E). In control cultures, all labeled fibers were directed towards the dorsal spinal cord and terminated there, where they normally enter the gray matter at E13 (Figure 6B). No axons were observed around the PBS-loaded control beads (Figure 6F). Axons that normally enter the gray matter through dorsal spinal cord grow towards the midline when presented with a localized NT-3 source, and new axon growth towards the NT-3 bead is initiated from DRGs, entering the spinal cord at ectopic loci at lateral mid-spinal cord (Figure 6G). NT-3 is therefore capable of acting as a chemoattractant for DRG 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