noc-18 and 2-phenyl-4-4-5-5-tetramethylimidazoline-1-oxyl-3-oxide

Asymmetric Ca(2+) signals across the growth cone mediate attractive or repulsive axon guidance depending on the occurrence of Ca(2+)-induced Ca(2+) release (CICR) through ryanodine receptors (RyRs). Although the neuronal isoform of nitric oxide (NO) synthase (nNOS) is highly expressed in developing dorsal root ganglion (DRG) neurons, the role of NO in axon guidance remains essentially unknown. Here we report that the NO-cGMP pathway negatively regulates CICR to control the directional polarity of DRG axon guidance. Intracellular levels of NO and cGMP depend on extracellular substrates: laminin activates the NO-cGMP pathway, whereas the adhesion molecule L1 does not. The activity of NO and cGMP determines the turning direction of growth cones with respect to asymmetric Ca(2+) signals that are produced by photolysing caged Ca(2+). The Ca(2+) signals cause growth cone repulsion on a laminin substrate, which is converted to attraction by pharmacological blockade of the NO-cGMP pathway or genetic deletion of nNOS. Conversely, Ca(2+)-induced growth cone attraction on an L1 substrate is converted to repulsion by increasing NO levels. Such NO-mediated switching of turning direction involves the regulation of CICR through RyRs. Furthermore, growth cone repulsion induced by an extracellular gradient of a physiological cue, neurotrophin-4, is dependent on Ca(2+) signals and converted to attraction by inhibiting the NO-cGMP pathway. These results suggest that, on contact with different adhesive environments, growth cones can change their turning responses to axon guidance cues by modulating CICR via endogenous NO and cGMP.

Topics: Animals; Calcium Signaling; Cell Polarity; Cells, Cultured; Chick Embryo; Cyclic AMP; Cyclic GMP; Cyclic N-Oxides; Cytosol; Egtazic Acid; Enzyme Inhibitors; Free Radical Scavengers; Ganglia, Spinal; Growth Cones; Imidazoles; Lasers; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Ryanodine Receptor Calcium Release Channel; Time Factors

In higher vertebrates, the central nervous system (CNS) is unable to regenerate after injury, at least partially because of growth-inhibiting factors. Invertebrates lack many of these negative regulators, allowing us to study the positive factors in isolation. One possible molecular player in neuronal regeneration is the nitric oxide (NO)-cyclic guanosine-monophosphate (cGMP) transduction pathway which is known to regulate axonal growth and neural migration. Here, we present an experimental model in which we study the effect of NO on CNS regeneration in flat-fillet locust embryo preparations in culture after crushing the connectives between abdominal ganglia. Using whole-mount immunofluorescence, we examine the morphology of identified serotonergic neurons, which send a total of four axons through these connectives. After injury, these axons grow out again and reach the neighboring ganglion within 4 days in culture. We quantify the number of regenerating axons within this period and test the effect of drugs that interfere with NO action. Application of exogenous NO or cGMP promotes axonal regeneration, whereas scavenging NO or inhibition of soluble guanylyl cyclase delays regeneration, an effect that can be rescued by application of external cGMP. NO-induced cGMP immunostaining confirms the serotonergic neurons as direct targets for NO. Putative sources of NO are resolved using the NADPH-diaphorase technique. We conclude that NO/cGMP promotes outgrowth of regenerating axons in an insect embryo, and that such embryo-culture systems are useful tools for studying CNS regeneration.

Topics: Animals; Axons; Cyclic GMP; Cyclic N-Oxides; Drug Interactions; Embryo, Nonmammalian; Free Radical Scavengers; Ganglia, Invertebrate; Guanylate Cyclase; Imidazoles; Indoles; Locusta migratoria; NADPH Dehydrogenase; Nerve Crush; Nerve Regeneration; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Organ Culture Techniques; Serotonin; Time Factors