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The central complex, a set of neuropils in the center of the insect brain, plays a crucial role in spatial aspects of sensory integration and motor control. Stereotyped neurons interconnect these neuropils with one another and with accessory structures. We screened over 5,000 Drosophila melanogaster GAL4 lines for expression in two neuropils, the noduli (NO) of the central complex and the asymmetrical body (AB), and used multicolor stochastic labeling to analyze the morphology, polarity, and organization of individual cells in a subset of the GAL4 lines that showed expression in these neuropils. We identified nine NO and three AB cell types and describe them here. The morphology of the NO neurons suggests that they receive input primarily in the lateral accessory lobe and send output to each of the six paired noduli. We demonstrate that the AB is a bilateral structure which exhibits asymmetry in size between the left and right bodies. We show that the AB neurons directly connect the AB to the central complex and accessory neuropils, that they target both the left and right ABs, and that one cell type preferentially innervates the right AB. We propose that the AB be considered a central complex neuropil in Drosophila. Finally, we present highly restricted GAL4 lines for most identified protocerebral bridge, NO, and AB cell types. These lines, generated using the split‐GAL4 method, will facilitate anatomical studies, behavioral assays, and physiological experiments. The authors use the multicolor flip‐out strategy in Drosophila to analyze neurons in two neuropils: the noduli of the central complex and the asymmetrical body. A set of split‐GAL4 lines is provided for previously published protocerebral bridge cell types as well as cell types presented here. [ABSTRACT FROM AUTHOR]

Left-right (LR) asymmetry of the brain is fundamental to its higher-order functions. The Drosophila brain's asymmetrical body (AB) consists of a structural pair arborized from AB neurons and is larger on the right side than the left. We find that the AB initially forms LR symmetrically and then develops LR asymmetrically by neurite remodeling that is specific to the left AB and is dynamin dependent. Additionally, neuronal ecdysone signaling inhibition randomizes AB laterality, suggesting that ecdysone signaling determines AB's LR polarity. Given that AB's LR asymmetry relates to memory formation, our research establishes AB as a valuable model for studying LR asymmetry and higher-order brain function relationships. [Display omitted] • The AB is first formed as a bilateral structure in the Drosophila brain • Neurite pruning occurs in the left, but not right, AB during AB's lateralization • Lateralization of the AB depends on dynamin in neurons • Neuronal ecdysone signaling polarizes left-right asymmetry of the AB The asymmetrical body (AB) is a left-right (LR)-asymmetric structure in the Drosophila brain. Sakamura et al. show that the AB is initially formed LR symmetrically and lateralized though neurite remodeling during the pupal stage. This remodeling depends on dynamin activity in neurons. Ecdysone signaling defines LR polarity of the AB. [ABSTRACT FROM AUTHOR]