Anatomical studies on the brain of the locust, Schistocerca gregaria: mapping of NADPH diaphorase and generation of a three-dimensional standard brain atlas

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 NADPHdpositive 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 NADPHdpositive 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. In order to understand the connectivity of neuronal networks, their constituent neurons are ideally studied in a common framework. Since morphological data from physiologically characterized and stained neurons usually arise from different individual brains, this can only be performed in a virtual standardized brain that compensates for interindividual variability. The desert locust, Schistocerca gregaria is an insect species used widely for the analysis of olfactory and visual signal processing, endocrine functions, and neural networks controlling motor output. To provide a common multi-user platform for neural circuit analysis in the brain of this species, we have generated a standardized 3D brain of the desert locust. Serial confocal images from wholemount locust brains were used to reconstruct 34 neuropil areas in ten brains. For standardization, we compared two different methods: an iterative shape averaging (ISA) procedure using affine transformations followed by iterative nonrigid registrations, and the Virtual Insect Brain (VIB) protocol, using global and local rigid transformations, followed by local nonrigid transformations. Both methods generated a standard brain, however for different applications. While the VIB technique was designed to visualize anatomical variability between the input brains, the purpose of the ISA method was the opposite, to remove this variability. A novel individually labeled neuron, connecting the lobula to the midbrain and deutocerebrum, was registered into the ISA atlas and demonstrates its usefulness and accuracy for future analysis of neural networks. The locust standard brain is accessible at www.3d-insectbrain.com