Behavioral neuroimaging in birds using PET

•A comprehensive set of neuroimaging methods to study bird behaviors is presented.•An enhanced 3D atlas of pigeon brain was developed and applied to study flight.•It is likely that F-18 FDG behaves as a reversible PET tracer in the bird brain. Background: Birds comprise the most diverse group of terrestrial vertebrates. This success likely is related to the evolution of powered flight over 75 mya. Modern approaches for studying brain function, however, have yet to be fully adapted and applied to birds, especially as they relate to specific behaviors including flight. New method: We have developed a comprehensive set of in vivo experimental methods utilizing PET imaging with F-18 labeled fluorodeoxyglucose (FDG) to study regional changes in metabolism specifically related to flight, yet applicable to other behaviors as well. It incorporates approaches for selection of species, behavioral/imaging paradigm, animal preparation, radiotracer injection route, image quantification, and image analysis via an enhanced brain atlas. We also carried out preliminary modeling studies to better understand tracer kinetics. Results: The methods were successful in identifying brain regions statistically associated with flight using only 8 animals. Peak brain uptake of FDG between birds and rodents is similar despite much higher blood glucose levels in birds. We also confirmed that brain uptake of FDG steadily decreases after the initial peak and provide evidence that it may be related to greater dephosphorylation of FDG phosphate than that observed in mammals. Comparison with existing methods: FDG PET has been used in only a few studies of the bird brain. We introduce a new species, more realistic flight behavior, paired (test/retest) design, and improved quantification and analysis approaches. Conclusions: The proposed imaging protocol is non-invasive yet sensitive to regional metabolic changes in the bird brain related to behavior.