R34 BRAIN Initiative. Optical interrogation of neural circuits in Manduca Project Summary / Abstract This BRAIN Circuits Planning Project will help to bridge the gap between well-established brain studies on the fruit fly, Drosophila, and complex behaviors exhibited more generally by other species. The immediate goal is to adapt and apply optogenetic technologies to identify brain circuits responsible for specific behaviors in the Tobacco Hawkmoth, Manduca sexta. In the long term, this will help to identify fundamental principles underlying brain function by comparing Manduca brain circuits with those underlying similar behaviors in Drosophila and other species. Manduca is an ideal organism for making these comparisons. For more than 50 years Manduca has been an important experimental species for understanding how nervous systems develop and how sensory signals in the environment are used to control behavior. It is a large insect that is easily raised in the laboratory and its behavior, anatomy, and physiology are known in detail. Furthermore, Manduca and Drosophila are superficially similar in their anatomy and development, making many comparisons relatively straightforward, but the two species also have distinct behavior and CNS organization. The experiments detailed in this project will demonstrate that optogenetic components can be expressed in Manduca and used to monitor and manipulate larval neural circuits and behavior. It will introduce new methods and improve on existing techniques of Manduca gene targeting and transgenesis. This will include testing promoters to drive expression of transgenes in central neurons, nociceptors and mechanosensors, validating their ability to activate or inhibit neuronal activity, and assessing effects on behavior. These experiments will prepare the groundwork for a BCP R01 project to identify the neural circuits that underlie nociceptive behaviors in Manduca larvae. In Drosophila, nociception involves multiple parallel circuits and the integration of different sensory modalities that are not fully understood. By comparing nociceptive pathways in these species, it is expected any common organizing principles for processing noxious or damaging stimuli will be identified. This will also inform studies of nociception and pain perception carried out in other species including vertebrates. In addition to enabling future work on nociception and locomotion, the tools developed here will be used by other researchers to study different behaviors and life stages, including flight control, object tracking, navigation, and multimodal integration.