Multiscale Optical Ca²⁺ Imaging of Tonal Organization in Mouse Auditory Cortex

Spatial patterns of functional organization, resolved by microelectrode mapping, comprise a core principle of sensory cortices. In auditory cortex, however,recent two-photon Ca²⁺ imaging challenges this precept, as the traditional tonotopic arrangementappears weakly organized at the level of individual neurons. To resolve this fundamental ambiguity about the organization of auditory cortex, we developed multiscale optical Ca²⁺ imaging of unanesthetized GCaMP transgenic mice. Single-neuron activity monitored by two-photon imaging was precisely registered to large-scale cortical maps provided bytranscranial widefield imaging. Neurons in theprimary field responded well to tones; neighboringneurons were appreciably cotuned, and preferred frequencies adhered tightly to a tonotopic axis. Bycontrast, nearby secondary-field neurons exhibited heterogeneous tuning. The multiscale imaging approach also readily localized vocalizationregions and neurons. Altogether, these findings cohere electrode and two-photon perspectives, resolve new features of auditory cortex, and offer apromising approach generalizable to any cortical area.