Data from: Specific expansion of motor cortical projections in a singing mouse

Data and analysis for Isko et al., 2026. Behavioral data and analysis as
well as MAPseq data and analysis included. Elucidating how modifications
in neural circuit architecture drive behavioral innovation remains a key
challenge in neuroscience and evolutionary biology. In mammals, the
neocortex is posited to play a critical role in facilitating rapid
behavioral innovations, such as language. Although changes in long-range
connectivity have been proposed to underlie such innovations, these
hypotheses largely remain untested quantitatively, partly due to the lack
of high-throughput neuronal projection data at single-neuron resolution
across species. To address this, we studied the Alston’s singing mouse
(Scotinomys teguina), which exhibits a striking vocal behavior absent in
the laboratory mouse (Mus musculus), to quantitatively determine
species-specific changes in motor cortical projections throughout the
brain. We used bulk tracing, serial two-photon tomography, and
high-throughput DNA sequencing of over 76,000 barcoded neurons to discover
a specific and substantial expansion (~3 fold) of orofacial motor cortical
(OMC) projections to the auditory cortical region (AudR) and the midbrain
periaqueductal gray (PAG), both implicated in vocal behaviors. Moreover,
analysis of individual OMC neurons’ projection motifs revealed
preferential expansion of exclusive projections to AudR in the singing
mouse. Our results suggest that selective expansion of ancestral motor
cortical projections may lead to behavioral divergence over short
timescales, allowing mechanistic investigations of enhanced cortical
control over vocalizations — a crucial preadaptation for human language.
This approach of comparing recently-diverged species with drastic
behavioral divergences can be readily generalized across other model
clades to discover quantitative rules of neural circuit evolution.