Imaging TR or CT neurons in A1 of awake mice. (A) Cartoon illustration of the auditory thalamocortical and corticothalamic circuits. (B) Schematic diagram of the injection site of AAV2/1-Cre in the MGB and AAV2/9-CaMKⅡ-DIO -GCaMP6s in AuC. (C) Coronal slice showing AAV-GCaMP6s expression in the AuC on day 21. (D) Fluorescence intensity of TR neurons in AuC with distance from pia (0%) to the L6/WM border (100%). (E) Schematic diagram of the injection site of CVS-GCaMP6s in the MGB. (F) Coronal slice showing the CVS-GCaMP6s retrogradely labeled neurons in the AuC on day 7. (G) Fluorescence intensity of CT neurons in AuC with distance from pia (0%) to the L6/WM border (100%). (H) Left: wide-field imaging in AAV-GCaMP6s-expressing mouse (TR neurons), fluorescence response to pure-tone stimulation with 4 kHz (blue area) and 32 kHz (red area). White dotted lines outline the A1, A2, and AAF boundaries. Right: wide-field imaging in CVS-GCaMP6s-expressing mouse (CT neurons). (I) Left: a fluorescent micrograph of a coronal slice after CTB-555 loading guided by 2P imaging into A1 in a CVS-GCaMP6s-expressing mouse. Right: Micrograph of a coronal slice of the MGB from the same mouse. CTB-555 retrogradely labeled neurons were mainly concentrated in the MGBv. (J) Left: wide-field imaging (in a GCaMP6s-expressing mouse) in A1, fluorescence response to pure-tone stimulation with 4 kHz (low-frequency (LF) area) and 32 kHz (high-frequency (HF) area), same abbreviation for all subsequent figures. Inset panels outlined by dashed boxes show the magnified views of 2P imaging of TR or CT neurons. Right: examples of 2P images of TR and CT neurons in vivo, respectively.

Tonotopic gradients of TR versus CT neurons. (A) Two-photon image of the focal plane used to image TR neurons in A1. The dashed circle indicates the tuned neurons. Fluorescence traces of neurons in the right panel are indicated by dashed circle ordered according to sound frequency and level. The five traces associated with the five repeats of each stimulus are plotted in gray. The average calcium signals are plotted in black. The red outline marks the BF response of the neuron. (B) Same as panel A) but for CT neurons in A1. (C) BF map of TR neurons. (D) BF map of CT neurons. (E) Schematic illustrating the recording locations of individual fields of view of TR neurons in A1. Outlines are color-coded according to the median BF response in the respective fields of view, with their BFs (kHz) color-coded by the scheme below. N = 23 focal planes, from 5 mice. (F) Same as panel E) but for CT neurons. N = 40 focal planes, from 10 mice. (G) BFmedian plotted against distance along the tonotopic axis for TR neurons. Scatter plots showing the correlation between the cellular BFmedian values measured in the fields of view with 2P imaging and the corresponding extrapolated brain surface frequencies determined by wide-field imaging of TR neurons. (H) Same as panel G) but for CT neurons. (I) Reconstruction of the relative spatial locations of tuned TR neurons that covered the whole A1 area and color-coded according to each neuron’s BF. N = 481 neurons from 5 mice. (J) Plots of TR neurons’ BF and their relative distances along the caudal-to-rostral axis. (K) Same as panel i but for CT neurons. N = 491 neurons from 10 mice. (L) Same as panel J) but for CT neurons. (M) Distribution histogram of BF from all neurons in the dataset (TR: N = 481 neurons; CT: N = 491 neurons). (N) Distribution histogram of BF neurons that were identified in the LF area by wide-field imaging (TR: N = 116 neurons; CT: N = 98 neurons). (O) Distribution histogram of BF neurons that were identified in HF area by wide-field imaging (TR: N = 91 neurons; CT: N = 90 neurons).

Heterogeneity of frequency preference in TR versus CT neurons. (A) Spatial distribution of all tone-selective neurons in an example imaging plane of TR neurons from LF (left) and HF (right) imaging area. (B) Same as panel A) but for CT neurons. BFs (kHz) color code is on panel A). (C) The comparison of field (200 µm) IQR of TR and CT. N = 23 focal planes from 5 mice for TR; N = 40 focal planes from 10 mice for CT. “TR”: 1.29\0.91–1.88 octaves (median\25–75% percentiles, same notation for all subsequent data), “CT”: 1.94\1.29–2.58 octaves. P = 0.016, two-sided Wilcoxon rank-sum test, *P < 0.05, **P < 0.01, and ***P < 0.001, same statistics for boxplots. (D) The comparison of the field IQR of TR and CT neurons in LF and HF areas. LF area: N = 4 focal planes from 3 mice for TR; N = 9 focal planes from 5 mice for CT. HF area: N = 5 focal planes from 5 mice for TR; N = 11 focal planes from 10 mice for CT. “TR: LF area”: 0.43\0.22–0.86 octaves, “TR: HF area”: 1.29\1.02–1.32 octaves, “CT: LF area”: 1.94\1.45–2.42 octaves, “CT: HF area”: 1.83\0.94–2.58 octaves. P (TR: LF area, HF area) = 0.11, P (CT: LF area, HF area) = 0.82, P (LF area: TR, CT) = 0.0084, P (HF area: TR, CT) = 0.25. (E) Cartoon showing how the local and minimum percentages were computed. (F) Cumulative percentage plot displaying local (25 µm radius) IQR around each neuron of TR and CT. Median local IQRBF of TR neurons = 0.86 octaves, N = 120 local planes from 5 mice; Median local IQRBF of CT neurons = 1.29 octaves; N = 105 local planes from 10 mice. (G) The comparison of the local (25 µm radius) IQR of TR and CT neurons in LF and HF areas. LF area: N = 34 local planes for TR; N = 20 local planes for CT. HF area: N = 23 local planes for TR; N = 16 local planes for CT. “TR: LF area”: 0.65\0.32–0.97 octaves, “TR: HF area”: 1.29\0.54–2.58 octaves, “CT: LF area”: 1.29\0.75–2.04 octaves, “CT: HF area”: 0.91\0.86–1.61 octaves. P (TR: LF area, HF area) = 0.0021, P (CT: LF area, HF area) = 0.69, P (LF area: TR, CT) = 0.0057, P (HF area: TR, CT) = 0.34. (H) Local IQRBF plotted against distance along the tonotopic axis for TR neurons. (I) The comparison of the minimum distance Δ Frequency of TR and CT neurons in LF and HF area. LF area: N = 116 paired neurons for TR; N = 87 paired neurons for CT. HF area: N = 91 paired neurons for TR; N = 78 paired neurons for CT. “TR: LF area”: 0.43\0.00–0.86 octaves, “TR: HF area”: 0.86\0.00–1.72 octaves, “CT: LF area”: 0.43\0.00–1.29 octaves, “CT: HF area”: 0.43 \0.00–1.29 octaves. P (TR: LF area, HF area) = 4.25e-04, P (CT: LF area, HF area) = 0.79, P (LF area: TR, CT) = 0.18, P (HF area: TR, CT) = 0.025. (J) Δ Frequency plotted against distance along the tonotopic axis for TR neurons.

Receptive field properties of TR versus CT neurons. (A) One representative FRA from a specific type of TR neuron. Types are V shape, I shape, and O shape. See text for further details. (B) One representative FRA from a specific type of CT neuron. (C) Comparison of fraction of neurons from V, I, and O shaped neurons in TR and CT. (D) Comparison of half-peak bandwidth of V-shaped TR and CT neurons. N = 190 neurons from 5 mice for TR; N = 249 neurons from 10 mice for CT. “TR”: 1.08\0.86–1.29 octaves, “CT”: 1.94\1.29–2.37 octaves. P = 4.86e-29. (E) Comparison of Q value (bandwidth of BF/half bandwidth) of V-shaped TR and CT neurons. N = 190 neurons from 5 mice for TR; N = 249 neurons from 10 mice for CT. “TR”: 2.00\1.50–2.29, “CT”: 1.78\ 1.50–2.00. P = 0.035. (F) Comparison of the Q value of V-shaped TR and CT neurons in LF and HF areas. LF area: N = 56 neurons for TR; N = 49 neurons for CT. HF area: N = 37 neurons for TR; N = 41 neurons for CT. “TR: LF area”: 1.78\1.60–2.00, “TR: HF area”: 2.00\1.58–2.43, “CT: LF area”: 1.78\1.50–2.22, “CT: HF area”: 1.60\1.33–1.88. P (TR: LF area, HF area) = 0.20, P (CT: LF area, HF area) = 0.032, P (LF area: TR, CT) = 0.88, P (HF area: TR, CT) = 0.0032. (G) Comparison of maximum bandwidth (BWmax) from V, I, and O shaped neurons in TR and CT. V shape: N = 190 neurons for TR, N = 249 neurons for CT; “TR”: 2.15\1.72–2.58 octaves, “CT”: 3.44\2.58–3.87 octaves; P = 4.18e-29. I shape: N = 174 neurons for TR, N = 112 neurons for CT; “TR”: 0.86\0.43–1.29 octaves, “CT”: 1.29\0.43–3.44 octaves; P = 4.66e-05. O shape: N = 117 neurons for TR, N = 130 neurons for CT; “TR”: 1.29\0.86–2.15 octaves, “CT”: 3.01\2.15–3.44 octaves; P = 4.30e-19. (H) Distribution histogram of BWmax from all neurons in the dataset. (I) Comparison of the BWmax of TR and CT neurons. N = 481 neurons from 5 mice for TR, N = 491 neurons from 10 mice for CT; “TR”: 1.29\0.86–2.15 octaves, “CT”: 3.01\2.15–3.87 octaves; P = 1.89e-51. (J) Comparison of the BWmax of TR and CT neurons in LF and HF areas. LF area: N = 116 neurons for TR; N = 98 neurons for CT. HF area: N = 91 neurons for TR; N = 90 neurons for CT. “TR: LF area”: 1.72\1.29–2.15, “TR: HF area”: 1.29\ 0.86–2.47, “CT: LF area”: 2.58\ 1.72–3.44, “CT: HF area”: 3.01\ 1.29–3.87. P (TR: LF area, HF area) = 0.034, P (CT: LF area, HF area) = 0.92, P (LF area: TR, CT) = 2.44e-08, P (HF area: TR, CT) = 2.28e-06. (K) Distribution of the monotonicity index (MI) of TR and CT neurons. (L) Comparison of the fraction of non-monotonic neurons of TR and CT. N = 23 focal planes from 5 mice for TR, N = 40 focal planes from 10 mice for CT; “TR”: 20.00%\12.92–31.49%, “CT”: 25.00%\14.58–40.00%; P = 0.33. (M) Comparison of the fraction of non-monotonic neurons of TR and CT in LF and HF areas. LF area: N = 4 focal planes from 3 mice for TR; N = 9 focal planes from 5 mice for CT. HF area: N = 5 focal planes from 5 mice for TR; N = 11 focal planes from 10 mice for CT. “TR: LF area”: 9.55%\6.08–13.50%, “TR: HF area”: 15.38%\14.66–29.38%; “CT: LF area”: 22.22%\17.59–31.67%, “CT: HF area”: 38.18%\16.67–60.00%; P (TR: LF area, HF area) = 0.032, P (CT: LF area, HF area) = 0.19, P (LF area: TR, CT) = 0.016, P (HF area: TR, CT) = 0.17. (N) Comparison of the MI of non-monotonic and monotonic neurons in TR and CT. Non-mon: N = 107 neurons for TR, N = 128 neurons for CT; “TR”: 0.42\0.33–0.47, “CT”: 0.35\0.26–0.44; P = 7.03e-04. Mon: N = 374 neurons for TR, N = 363 neurons for CT; “TR”: 0.78\0.63–1.00, “CT”: 1.00\0.73–1.00; P = 6.19e-11. (O) Comparison of the BF between monotonic and non-monotonic neurons in TR and CT. TR: N = 107 neurons for non-mon, N = 374 neurons for mon; “non-mon”: 6.60\2.93–12.10 kHz, “mon”: 8.90\3.60–16.30 kHz; P = 0.69. CT: N = 128 neurons for non-mon, N = 363 neurons for mon; “non-mon”: 8.90\3.60–19.15 kHz, “mon”: 6.60\2.70–12.10 kHz; P = 1.64e-04. (P) Comparison of the BWmax between monotonic and non-monotonic neurons in TR and CT. TR: N = 107 neurons for non-mon, N = 374 neurons for mon; “non-mon”: 1.72\0.86–2.15 octaves, “mon”: 1.29\0.86–2.15 octaves; P = 0.92. CT: N = 128 neurons for non-mon, N = 363 neurons for mon; “non-mon”: 3.44\2.15–3.87 octaves, “mon”: 3.01\2.15–3.87 octaves; P = 0.052.

Chronic in vivo 2P imaging of CT neurons. (A) Calcium imaging from L6 CT neurons in a CVS-expressing mouse, again with repeated imaging of the same focal planes on day 7 and 13 after virus injection. The dashed circle indicates the example neurons, showing fluorescence traces of neurons in the lower panel. (B) The comparison of the fraction of active CT neurons on day 7 and 13. N = 14 focal planes from 5 mice. “Day 7”: 79.14\59.26–86.11%, “Day 13”: 75.33\62.07–86.11%; P = 0.96, two-sided Wilcoxon signed-rank test, *P < 0.05, **P < 0.01, and ***P < 0.001, same statistics for boxplots. (C) The comparison of fraction of reliable CT neurons on day 7 and 13. “Day 7”: 12.70\9.09–26.67%, “Day 13”: 13.19\7.69–20.00%; P = 0.53. The definitions of “active” and “reliable” neurons see METHOD DETAILS.

Pie charts showing the percentage of responsive neurons to pure-tone stimulation of TR and CT neurons. N = 5 animals for TR neurons, N = 10 animals for CT neurons. The definitions of ‘‘silent’’ and ‘‘irregular’’ neurons see METHOD DETAILS.