Extended Data Figure 3. Two-photon calcium imaging of auditory cortical responses to pup calls and pure tones.

a-c, Neuropil correction on example data sets (N=1 region containing excitatory neurons; N=1 region containing inhibitory neurons). a, Top, correction was performed by measuring background fluorescence in the neuropil (‘NP’) surrounding each ROI (green) and local vasculature (‘V’). Bottom, example ΔF/F traces from an excitatory neuron before and after correction (see methods). b,c, Neuropil correction had no significant effect on prototype-evoked ΔF/F (%) in excitatory neurons (b, n=52 neurons, p=0.57) or inhibitory neurons (c, n=64 neurons, p=0.14; two-tailed unpaired Kolmogorov-Smirnov test). d, Example ΔF/F traces from three neurons acquired at 16 Hz; colored number=ΔF/F (%). Responses from this experienced virgin were consistent with neuronal responses acquired at 4 Hz (Fig. 1e). e, Percentage of prototype-responsive excitatory neurons in experienced (N=9 mice) and naive virgins (N=12; p=0.74; two-tailed unpaired t-test). f, Example heatmaps of prototype-responsive neurons from an experienced (left) and naive virgin (right). g,h, Raw neuronal tuning from experienced (N=9) and naive virgins (N=12) summarized in Fig. 1f. i-m, Tuning to temporally-modulated tone sequences. i, Example stimulus set: five sequential tone pips (e.g., 32 kHz, 80 ms) with the following ISIs: 75, 175, 375, or 575 ms (ISI bin ±25 ms). j, Left, example ΔF/F traces evoked by temporally-modulated sequences of 32 kHz tones. Right, sample cell quantification. ΔF/F (%) normalized to the prototypical stimulus. k, Tuning width (normalized ΔF/F averaged across all stimuli). Experienced: N=3 mice, n=94 neurons; naive: N=4, n=45; p=0.97 (unpaired two-tailed t-test). l, Sample imaging region from a naive virgin. Prototypical calls and temporally-modulated 32 kHz tones with ISIs ~175 ms (5 Hz) activated a subset of the same cells (green). These neurons could have distinct temporal tuning to ISIs (inset). m, We observed higher normalized ΔF/Fs to temporally-modulated tones (N=5 mice) than to pup call morphs (n=11–12) in naive auditory cortex (75 ms, p=0.009; 375 ms, p=0.04; 575 ms: p=0.004; unpaired two-tailed Mann-Whitney test). Median±interquartile. All data shown are mean±s.e.m. except (m); Stats: *p<0.05, **p<0.01