Fig. 1: Holistic bursting (HB) cells emerged from quiescence in a stepwise manner through learning.

a Sound-licking associative learning task (left) and learning curve (right). Chord 1 (2.0, 2.7, 3.6, 4.9 kHz) associated with water from right side; chord 2 (8.9, 12.1, 16.3, 22.0 kHz) associated with water from left side. Data with error bars are presented as the mean ± sem. Credits of Ms. Jia Lou. b Left: Two-photon image of an example neuron in AuC L2/3 labeled with GCaMP6m (cropped from whole image, scale bar: 10 μm, same arrangements for all example cells hereinafter). Right: Sound-evoked Ca²⁺ signals of the example neuron day by day (trial-averaged traces in black overlayed with single trials in gray). 33 neurons of 1862 imaged neurons in 13 mice. were repeated independently with similar results. c Simultaneous two-photon Ca²⁺ imaging and loose-patch recording of the same example neuron in panel b on day 6. Upper: Ca²⁺ signals and corresponding loose-patch responses in 5 consecutive trials. Lower: magnified view of the loose-patch recordings. Red sections of traces identified as bursting events (same hereinafter). Purple shade: sound stimulation, blue shade: water dispense, same for all figures hereinafter. 2PM: two-photon microscope. Credits of Ms. Jia Lou. d Left: Targeted loose-patch recordings of a neuron that emerged with high Ca²⁺ responsiveness to chord 2 through learning. Right: Box-whisker plots (boxes: median & 25–75% percentiles, whiskers: 10–90% percentiles, same convention applies to all boxplots hereinafter) 12\5.5–18 spikes per chord response, 4\1–6 per sum of 4 constituent tones response. N = 22 trials (pooled, 4 HB cells recorded in 3 mice), ****P = 2.2e−05, t₂₁ = 5.437, Two-sided paired t-test. e A session of loose-patch recording of an example HB cell including consecutive sound-evoked response events. 235 events pooled from 14 cells were repeated independently with similar results. f A session of loose-patch recording of an example non-HB cell, spontaneous activity in the absence of sound stimuli. 79 events pooled from 9 cells were repeated independently with similar results. g No significance of spontaneous burst events rate (1/s) between non-HB cells and HB cells. Burst firing rate: non-HB cells, 0.04\0.02–0.06 Hz; HB cells, 0.025\0–0.0775 Hz. n.s., P = 0.7550, Mann–Whitney U: 552, 9 non-HB cells in 5 mice, 14 HB cells in 10 mice. Two-sided Mann-Whitney test. h Distribution of sound-evoked burst firing probability of HB cells. 100%\87.5–100% (median\25–75% percentiles), 14 HB cells in 10 mice. i Violin plots of number of spikes per burst event among non-HB cell spontaneous burst events, HB cell spontaneous burst events, and HB cell sound-evoked burst events. Non-HB spon. vs. HB spon., n.s, P = 0.3213; Non-HB spon. vs. HB evoked, ****P = 7.5e−11; HB spon. vs. HB evoked, ****3.1e−5, F _{(2, 371)} = 28.18; One-way anova test and Tukey post-test. j Violin plots of instaneous firing rate (Hz) within burst event among non-HB cell spontaneous burst events, HB cell spontaneous burst events, and HB cell sound-evoked burst events. Non-HB spon. vs. HB spon., n.s, P = 0.4241; Non-HB spon. vs. HB evoked, ****P = 3.8e−5; HB spon. vs. HB evoked, *P = 0.0344; F _{(2, 372)} = 10.99; One-way anova test and Tukey post-test. k Left: sound-evoked (chord 2) Ca²⁺ signals of example cells emerged from quiescence to bursting (high Ca²⁺, Δf/f > 0.8) responsiveness through learning. Right: summary of Ca²⁺ responses before and after the transition for the quiescence-emerged cells. Chord 1, Δf/f value before transition: 0.48\0.23–0.60, after transition, 1.02\0.85–1.90, N = 13 cells, ***P = 2.7e−04, t₁₂ = 5.082; Chord 2, Δf/f value before transition: 0.34\0.26–0.46, after transition, 0.97\0.84–1.47, N = 20 cells. ****P = 1.1e−7, t₁₉ = 8.242, Two-sided paired t-test. Box-whisker plots: boxes represent 25% and 75% percentiles (Q1 and Q3), central bars represent the median, whiskers represent Q1−1.5 × IQR (interquartile range) and Q3 + 1.5 × IQR. l Stacked histograms showing the number of cells exhibited bursting responsiveness in each training day for all cells tagged as bursting on day 6. Data pooled from altogether 1862 imaged neurons in 13 mice.