Brain-wide projection reconstruction of single functionally defined neurons

Reconstructing axonal projections of single neurons at whole-brain level is a currently converging goal of the neuroscience community, which is fundamental to understanding the logic of information flow in the brain. Thousands of single neurons from different brain regions recently have been morphologically reconstructed, but the corresponding physiological functions of these reconstructed neurons are lacking. By combining two-photon Ca2+ imaging with targeted single-cell plasmid electroporation, we reconstructed the brain-wide morphologies of single neurons that were defined by a sound-evoked response map in the auditory cortex of awake mice. Long-range interhemispheric projections can be reliably labelled with co-injection of adeno-associated virus. This method avoids the randomness and ambiguity of conventional methods of neuronal morphological reconstruction, offering an avenue for developing a precise one-to-one map of neuronal projection and physiological function. Our method can be readily implemented in many laboratories that have been equipped with a standard two-photon microscope and electrophysiological devices.