Neural imaging in songbirds using fiber optic fluorescence microscopy

The song control system of juvenile songbirds is an important model for studying the developmental acquisition and generation of complex learned vocal motor sequences, two processes that are fundamental to human speech and language. To understand the neural mechanisms underlying song production, it is critical to characterize the activity of identified neurons in the song control system when the bird is singing. Neural imaging in unrestrained singing birds, although technically challenging, will advance our understanding of neural ensemble coding mechanisms in this system. We are exploring the use of a fiber optic microscope for functional imaging in the brain of behaving and singing birds in order to better understand the contribution of a key brain nucleus (high vocal center nucleus; HVC) to temporal aspects of song motor control. We have constructed a fluorescence microscope with LED illumination, a fiber bundle for transmission of fluorescence excitation and emission light, a similar to 2x GRIN lens, and a CCD for image acquisition. The system has 2 mu m resolution, 375 mu m field of view, 200 mu m working distance, and 1 mm outer diameter. As an initial characterization of this setup, neurons in HVC were imaged using the fiber optic microscope after injection of quantum dots or fluorescent retrograde tracers into different song nuclei. A Lucid Vivascope confocal microscope was used to confirm the imaging results. Long-term imaging of the activity of these neurons in juvenile birds during singing may lead us to a better understanding of the central motor codes for song and the central mechanism by which auditory experience modifies song motor commands to enable vocal learning and imitation.