PROJECT SUMMARY / ABSTRACT - Overall The Berghia Brain Project is a multi-institution collaborative project to examine fundamental issues about how brain circuits interact with distributed networks. There is a growing awareness of the importance of peripheral networks to human health, but it is difficult to study how such distributed networks interact at a cellular level with the brain in mammals. This project focuses on the nudibranch mollusc, Berghia stephanieae because it provides a unique opportunity to challenge fundamental concepts in neuroscience. The project examines the dynamics, network structure, algorithms, and neural implementation of behaviors related to mechanosensation, olfaction, and vision. The project addresses the critical need for phylogenetic diversity in neuroscience by developing genetic tools and protocols to make Berghia an accessible laboratory species for neuroscience. Berghia has a small brain with fewer than 5000 neurons in the adult and one tenth that number in juveniles. Understanding the impact of neurogenesis on functioning networks has implications for human memory circuits, which also gain neurons. In addition, Berghia has over 9000 neurons in each of its olfactory (rhinophore) ganglia and untold numbers of neurons that form a sub-epidermal nerve net on the animal?s body. The project will create a multi- feature, multi-scale atlas of neurons, which will feature connectomes of the adult brain and the whole body of a juvenile obtained from serial reconstruction of electron micrographs. These connectomes will be aligned with fluorescence images of neuronal gene expression and immunohistochemistry. The project will test solutions to automatically segment images and rapidly create wiring diagrams. Neurons will be further identified through cell- lineage tracing to determine the origins of identified neurons and classes of other neurons. Analysis of single neuron RNA sequencing will provide new insights into circuitry and neural identity. Expansion microscopy will allow a rich accounting of peripheral neurons. The activity of neurons in the peripheral net and the brain will be optically recorded with voltage- and calcium-sensitive dyes and genetically-encoded sensors to image information flow in response to mechanical, olfactory, and visual stimuli. The project will test standard hypotheses related to how these systems work in a non-standard organism with the goal of determining the generality of canonical concepts such as somatotopic mapping of the body, glomerular organization of olfactory systems, and modular organization of vision.