Cellular and widefield imaging of sound frequency organization in primary and higher-order fields of the mouse auditory cortex

The mouse auditory cortex (ACtx) contains two core fields – A1 and AAF – arranged in a mirror reversal tonotopic gradient. The best frequency (BF) organization and naming scheme for additional higher-order fields remain a matter of debate, as does the correspondence between smoothly varying global tonotopy and heterogeneity in local cellular tuning. Here, we performed chronic widefield and 2-photon calcium imaging from the ACtx of awake Thy1 -GCaMP6s reporter mice. Data-driven parcellation of widefield maps identified five fields, including a previously unidentified area at the ventral posterior extreme of the ACtx (VPAF) and a tonotopically organized suprarhinal auditory field (SRAF) that extended laterally as far as ectorhinal cortex. Widefield maps were stable over time, where single pixel BFs fluctuated by less than 0.5 octaves during a one-month imaging period. After accounting for neuropil signal and frequency tuning strength, BF organization in neighboring layer 2/3 neurons was intermediate to the heterogeneous salt and pepper organization and the highly precise local organization that have each been described in prior studies. Multiscale imaging data suggest there is no ultrasonic field or secondary auditory cortex in the mouse. Instead, VPAF and a dorsoposterior field (DP) emerged as the strongest candidates for higher-order auditory areas.