To study the general anatomy of the monarch brain, we first performed find more three-dimensional

reconstructions of the major brain neuropils. We labeled whole-brain preparations of the monarch with antibodies against the synaptic marker Synapsin, acquired confocal image stacks, and, based on these images, reconstructed the size and shape of identifiable brain regions. The resulting architecture of the monarch brain (Figures 2A–2C) was similar to that of other insect brains, with close resemblance to the brain of the hawkmoth Manduca sexta, the only other lepidopteran brain that has been examined in comparable detail ( El Jundi et al., 2009). In the monarch optic lobes, we identified and reconstructed the medulla, the lobula, the lobula plate, and the accessory medulla. In the central brain, we identified and reconstructed the central complex (CC), the anterior optic tubercles (AOTu), the antennal lobes, and the mushroom bodies (Figures 2A–2C). Because the present work focused on sun compass neuropils, the mushroom body lobes were not separated into their components; higher image resolution is required to resolve their compressed and intertwined organization. The monarch AOTu, the first processing stage for sun compass information in the

central brain of the locust (Pfeiffer et al., 2005), can be divided into three components: the large upper division, the lower division, and the nodular division, with its distinct, glomerular organization (Figure 2D). With the AZD5363 staining procedure used,

the monarch LALs, the second relay stage for compass information in the locust brain, did not show distinct boundaries, consistent with the situation in the hawkmoth. Etomidate However, the monarch LALs were defined on both sides of the CC using single-cell morphologies (Figures 3C, 3E, and 3G). Positioned posterior of the antennal lobe, they extended anteriorly, approximately from the depth of the lower division of the central body (CBL), until they almost reached the anterior surface of the brain. Simultaneous dye-fills of multiple CBL-tangential neurons revealed a distinct neuropil region containing the postsynaptic endings of these cells (Figures 3A and 3B). Because of the typical, microglomerular shape of these endings, this neuropil region is probably the monarch homolog of the combined lateral triangle/medial olive areas of the locust (data not shown). Spatially, these areas were located on either side of the midline, between the LAL and the posterioventral surface of the mushroom body lobes, and slightly anterior to the CBL. We next focused on the detailed reconstruction of the monarch CC, the proposed integration site of sun compass information in the insect brain.