Multiphon imaging for understanding social brain function in tadpoles

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Project Summary Mother-infant bonding is a key relationship that lays a foundation for wellness throughout life. Social recognition is an important component of this relationship, as infants imprint on the smell of their mothers and use olfaction to distinguish their mother from others. This behavior is widespread in mammals, although the complexity of the mammalian brain and paucity of tools and repeated imaging capabilities to study neonate pups make it difficult to obtain a precise mechanistic understanding of the basic brain mechanisms for mother-infant bonding and communication in young. Mother-infant recognition and bonding also occur in other taxa, suggesting there are other species in which to study generalizable neural principles of parent-offspring interactions. Recently our labs have developed (1) poison frog tadpoles as a model to study social brain development and (2) multiphoton imaging approaches to enable in vivo recording of pigmented aquatic larvae. We propose to develop multiphoton in vivo imaging approaches to study the encoding of maternal odors in a social poison frog tadpole that can distinguish their mother from strangers. We hypothesize that the olfactory response landscape changes throughout development as tadpoles learn the smell of their mothers. We predict that a tadpole’s ability to distinguish their mother from strangers coincides with an increase in olfactory cells that fire with specificity to maternal odors, measured by multiphoton imaging of olfactory system neural activity repeatedly across development. Prior to these experiments, we will evaluate the performance of various multiphoton imaging techniques for optically accessible depth inside the brain of multiple species of poison frog tadpoles with varying levels of pigmentation. We will validate the results with rigorous statistical analyses and comparison of neural activity data with immunohistological imaging of brain slices and activity-dependent sequencing of olfactory sensory neurons. Understanding how amphibians learn and encode individual conspecific identity will either reveal alternative mechanisms of encoding olfactory-based recognition or which patterns of olfactory encoding are ancestral or generalizable features of vertebrate olfactory processing. Importantly, our approach will result in the development of multiphoton approaches for whole brain imaging of pigmented aquatic animals, which is a valuable toolkit of broad use for the neuroscience community. Successful completion of this project will allow us to obtain proof-of-principle data for proposing in vivo imaging of tadpole brains throughout development, in a comparative context, and within the framework of ethologically relevant behaviors, which is crucial for future R01 applications. Furthermore, establishing a recording protocol in tadpoles will allow for other aspects of neural function in amphibians, a research area that has thus far been limited due to technological constraints. In summary, our proposed research will help elucidate core olfactory imprinting principles in a relatively simple tadpole brain and reveal how these principles for olfactory encoding may generalize across vertebrate taxa.