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Localization of nitric oxide synthase in the central complex and surrounding midbrain neuropils of the locust Schistocerca gregaria

Nitric oxide (NO), generated enzymatically by NO synthase (NOS), acts as an important signaling molecule in the nervous systems of vertebrates and invertebrates. In insects, NO has been implicated in development and in various aspects of sensory processing. To understand better the contribution of NO signaling to higher level brain functions, we analyzed the distribution of NOS in the midbrain of a model insect species, the locust Schistocerca gregaria, by using NADPH diaphorase (NADPHd) histochemistry after methanol/formalin fixation; results were validated by NOS immunohistochemistry. NADPHd yielded much higher sensitivity and resolution, but otherwise the two techniques resulted in corresponding labeling patterns throughout the brain, except for intense immunostaining but only weak NADPHd staining in median neurosecretory cells. About 470 neuronal cell bodies in the locust midbrain were NADPHd‐positive positive, and nearly all major neuropil centers contained dense, sharply stained arborizations. We report several novel types of NOS‐expressing neurons, including small ocellar interneurons and antennal sensory neurons that bypass the antennal lobe. Highly prominent labeling occurred in the central complex, a brain area involved in sky‐compass orientation, and was analyzed in detail. Innervation by NOS‐expressing fibers was most notable in the central body upper and lower divisions, the lateral accessory lobes, and the noduli. About 170 NADPHd‐positive neurons contributed to this innervation, including five classes of tangential neuron, two systems of pontine neuron, and a system of columnar neurons. The results provide new insights into the neurochemical architecture of the central complex and suggest a prominent role for NO signaling in this brain area. J. Comp. Neurol. 484:206–223, 2005. © 2005 Wiley‐Liss, Inc.