Cellular and Widefield Imaging of Sound Frequency Organization in Primary and Higher Order Fields of the Mouse Auditory Cortex

Abstract

The mouse auditory cortex (ACtx) contains two core fields—primary auditory cortex (A1) and anterior auditory field (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 two-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 throughout a 1-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 dorsal posterior (DP) field emerged as the strongest candidates for higher order auditory areas.

Complexity of frequency receptive fields predicts tonotopic variability across species

“…Issa et al 18 reported more homogenous tonotopy in mouse A1 than other two-photon imaging studies 16, 17, 19, 20, 26 , although the local variability in BF was still higher than that of marmosets 31 . Two distinguishing methodological features of Zeng et al 31 and Issa et al 18 are: (1) Issa et al imaged tone responses in awake animals, and (2) neither study applied neuropil corrections.…”

“…In contrast to single- and double-peaked neurons, only 30% of complex neurons in a given imaging field were within the expected BF range, and their signal correlations were lower and less spatially dependent. Romero et al have recently shown that BFs are more locally variable among neurons with less well-defined frequency tuning 20 . Here, we further show that BFs are also more variable among nearby neurons with complex frequency receptive fields, even when their trial-to-trial responses at BF are equally reliable (as shown by our Fano Factor analyses).…”

“…Two distinguishing methodological features of Zeng et al 31 and Issa et al 18 are: (1) Issa et al imaged tone responses in awake animals, and (2) neither study applied neuropil corrections. Both two-photon calcium imaging 20, 26 and single unit electrophysiological studies 14 have demonstrated that there is little change in a neuron’s BF between passively listening and anaesthetized states, so this alone is unlikely to account for the smooth tonotopy observed by Issa et. al.…”

“…The results of such experiments in mouse A1 over the past decade have challenged our understanding of the cortical tonotopic map. While previous electrophysiological and large-scale imaging reports confirmed an overall tonotopic organization in mouse A1 13–15 , most two-photon imaging studies have shown that, at the more local scale, neighboring neurons vary widely in their frequency preferences 16–20 . Similarly, two-photon imaging has demonstrated local heterogeneity in whisker selectivity in mouse primary somatosensory cortex 21, 22 , and in orientation tuning in mouse primary visual cortex (V1) 23 .…”

“…Throughout the ascending auditory pathway, neurons progressively integrate spectral and temporal features of sound 32 , and the receptive fields of many A1 neurons are poorly predicted by a model of linear tuning to a single sound frequency 33, 34 . Recent studies have shown that the preferred frequencies of weakly tuned A1 neurons are poorly mapped 20, 26 . An outstanding question we address here is how neurons with reliable tuning to multiple frequency peaks impact on tonotopic organization.…”