Abstract
We followed the development of the nitric oxide‐cyclic guanosine monophosphate (NO‐cGMP) system during locust embryogenesis in whole mount nervous systems and brain sections by using various cytochemical techniques. We visualized NO‐sensitive neurons by cGMP immunofluorescence after incubation with an NO donor in the presence of the soluble guanylyl cyclase (sGC) activator YC‐1 and the phosphodiesterase‐inhibitor isobutyl‐methyl‐xanthine (IBMX). Central nervous system (CNS) cells respond to NO as early as 38% embryogenesis. By using the NADPH‐diaphorase technique, we identified somata and neurites of possible NO‐synthesizing cells in the CNS. The first NADPH‐diaphorase‐positive cell bodies appear around 40% embryogenesis in the brain and at 47% in the ventral nerve cord. The number of positive cells reaches the full complement of adult cells at 80%. In the brain, some structures, e.g., the mushroom bodies acquire NADPH‐diaphorase staining only postembryonically. Immunolocalization of L‐citrulline confirmed the presence of NOS in NADPH‐diaphorase‐stained neurons and, in addition, indicated enzymatic activity in vivo. In whole mount ventral nerve cords, citrulline immunolabeling was present in varying subsets of NADPH‐diaphorase‐positive cells, but staining was very variable and often weak. However, in a regeneration paradigm in which one of the two connectives between ganglia had been crushed, strong, reliable staining was observed as early as 60% embryogenesis. Thus, citrulline immunolabeling appears to reflect specific activity of NOS. However, in younger embryos, NOS may not always be constitutively active or may be so at a very low level, below the citrulline antibody detection threshold. For the CNS, histochemical markers for NOS do not provide conclusive evidence for a developmental role of this enzyme. J. Comp. Neurol. 518:1157–1175, 2010. © 2010 Wiley‐Liss, Inc.