Single-nuclei RNA sequencing of HVC and RA in zebra finches

The evolution of the six-layered neocortex is often credited with an increased capacity for complex behaviors and cognition in humans and other mammals. However, birds, despite lacking a laminated neocortex, display complex and non-instinctual behaviors including vocal learning, tool use, and problem-solving1,2. The evolution of brain circuits and cell-types supporting advanced behavioral repertoires remains poorly understood. Here in songbirds, we use single-cell RNA-sequencing to characterize the molecular identities of cells in the song motor pathway, a pallial circuit with function and connectivity that has been likened to the mammalian neocortex1,2. We find that each song region contains different glutamatergic excitatory projection neurons but similar sets of GABAergic inhibitory interneurons, similar to patterns of neuronal diversity in mammals and reptiles3,4. Song motor pathway glutamatergic neurons have gene expression patterns similar to those described in neocortical projection neurons, but at the level of transcription factor expression, display stronger similarity to neurons in the ventral pallium. We observed multiple GABAergic neuron classes that are conserved across amniotes, yet the most abundant class strongly resembles a cell-class that in mammals is not found in the neocortex but is present in non-neocortical pallial regions3,4. Thus, although pallial song-control regions contain neurons with similar molecular profiles to neocortical neurons, these pallial areas have a regional identity distinct from neocortex, indicating that complex behaviors such as vocal learning have evolved through the use of different brain circuits under similar functional constraints. Overall design: We carried out single-nuclei RNA-sequencing of zebra finch HVC and RA. Multiple individuals were pooled within each library.