Brain-wide projection reconstruction of single functionally defined neurons

Reconstructing axonal projections of single neurons at the whole-brain level is currently a converging goal of the neuroscience community that is fundamental for understanding the logic of information flow in the brain. Thousands of single neurons from different brain regions have recently been morphologically reconstructed, but the corresponding physiological functional features of these reconstructed neurons are unclear. By combining two-photon Ca²⁺ imaging with targeted single-cell plasmid electroporation, we reconstruct the brain-wide morphologies of single neurons that are defined by a sound-evoked response map in the auditory cortices (AUDs) of awake mice. Long-range interhemispheric projections can be reliably labelled via co-injection with an adeno-associated virus, which enables enhanced expression of indicator protein in the targeted neurons. Here we show that 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 patterns and physiological functional features.