The pattern of axonal projections early in the development of the nervous system lacks the precision present in the adult. During a developmental process of refinement, mistargeted projections are eliminated while correct projections are retained. Previous studies suggest that during development nitric oxide (NO) is involved in the elimination of mistargeted retinal axons, whereas brain-derived neurotrophic factor (BDNF) may stabilize retinal axon arbors. It is unclear whether these neuromodulators interact. This study showed that NO induced growth cone collapse and retraction of developing retinal axons. This effect was not attributable to NO-induced neurotoxicity. BDNF protected growth cones and axons from the effects of NO. This effect was specific to BDNF, because neither nerve growth factor (NGF) nor neurotrophin-3 (NT-3) prevented NO-induced growth cone collapse and axon retraction. Exposure to both BDNF and NO, but not either factor alone, stabilized growth cones and axons. Stabilized axons exhibited minimal retraction or extension. This response appears to be a new axon "state" and not simply a partial amelioration of the effect of NO, because lower doses of BDNF or NO allowed axon extension. Furthermore, BDNF/NO-induced growth cone stabilization correlated with the appearance of a cytochalasin D-resistant population of actin filaments. BDNF protection from NO likely was mediated locally at the level of the growth cone, because growth cones or individual filopodia in contact with BDNF-coated beads were protected from NO-induced collapse. These findings suggest a cellular mechanism by which some axonal connections are stabilized and some are eliminated during development.

A transient ipsilateral retinotectal projection is normally eliminated during embryonic development of the chick visual system. Administration of the NMDA receptor antagonist 5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) during the developmental period in which this projection normally disappears prevented its complete elimination. Previous studies showed that tectal cells express nitric oxide synthase during development, and blocking synthesis of nitric oxide also prevented elimination of the ipsilateral retinotectal projection. The effect of NMDA receptor blockade on nitric oxide synthase activity in tectal cells was assessed biochemically in chick embryos. Increasing concentrations of MK-801 resulted in a dose-dependent decrease in nitric oxide synthase activity. This result suggests that NMDA receptor activation can regulate nitric oxide synthase activity in the tectum. The degree of rescue of the ipsilateral retinotectal projection was compared in embryos treated either with MK-801 or with an inhibitor of nitric oxide synthesis, Nomega-nitro-L-arginine (L-NoArg). At comparable levels of inhibition of nitric oxide synthesis, no significant difference was observed in the degree of rescue mediated by NMDA receptor blockade or nitric oxide synthesis blockade. These results suggest that NMDA receptor-mediated elimination of the ipsilateral retinotectal projection is completely mediated via nitric oxide.

Growing axons during development are guided to their targets by the activity of their growth cones. Growth cones integrate positive and negative guidance cues in deciding the direction in which to extend. We demonstrated previously that treatment of embryonic retinal ganglion cells with brain-derived neurotrophic factor (BDNF) protects their growth cones from collapse induced by nitric oxide (NO). BDNF stabilizes growth-cone actin filaments against NO-induced depolymerization. In the present study, we examined the signaling mechanism involved in BDNF-mediated protection. We found that BDNF causes transient activation of protein kinase A (PKA) during the first 5 min of treatment. Treatment with PKA inhibitors before or in conjunction with BDNF treatment blocked the protective effects of BDNF. The effects of BDNF, however, were not blocked when addition of PKA inhibitors was delayed as little as 15 min after BDNF treatment. When cultures raised overnight in BDNF were treated with PKA inhibitors, BDNF-mediated protection did not end, demonstrating that the maintenance of the protective effects of BDNF is independent of PKA activity. The BDNF-induced activation of PKA was required for BDNF-mediated stabilization of growth-cone actin filaments against depolymerization by cytochalasin D. Finally, the initiation and maintenance of the protective effects of BDNF required protein synthesis. Collectively, these data demonstrate that PKA signaling is required only for an early phase of BDNF-mediated protection from NO-induced growth-cone collapse.

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