Extended Data Fig. 8: Validation of the viral construct labeling inhibitory neurons and additional analyses of the microablation-induced effects on excitatory and inhibitory neurons.

a. Representative example of colocalization between mDlx enhancer-driven tagBFP expression and Gad67. Scale: 100 μm. b. Quantification of colocalization. n = 7 cortical slices from two mice. The middle line, the bottom and top edges of the box plots indicate median, 25th and 75th percentiles, respectively, and the whiskers are the most extreme data points. The fraction of interneurons labeled with the blue fluorescent protein marker out of the entire quality qualified neurons was around 10% for each cohort (10.5 ± 0.5% (mean ± s.e.m. across mice) in sound responsive ablation, 9.8 ± 0.6% in non-sound responsive ablation, and 7.8 ± 0.4% in control, respectively), in line with previous works with auditory cortex in mature adult mice⁸². When we revisited the profile of cell types in microablated neurons, nearly all the ablated neurons were excitatory neurons (92.3 ± 2.0% for sound-responsive ablated neurons, 98.4 ± 0.9% for non-sound responsive ablated neurons), the fraction of which is higher than the average fraction of excitatory neurons in the population. c. Exemplary tuning curve of inhibitory neuron. d. Representative excitatory (left) and inhibitory (right) neurons, which change response amplitude to sound stimuli from day 5 to day 11, in sound responsive ablation. e. Baseline-normalized ratio of the total sound-evoked activity in excitatory and inhibitory neurons (see Methods). Two-sided t-test with FDR adjusted p-values between experimental cohorts on post-ablation days, * p < 0.05.; the normalized ratio of best amplitudes on day 15, 1.09 ± 0.088 for sound responsive ablation, 1.10 ± 0.11 for non-sound responsive ablation. f. Traces of detected calcium transients of an inhibitory neuron over days. We verified that the changes of response amplitude in inhibitory neurons after microablation (Fig. 5e) were not due to another feature like Ca²⁺ kinetics in somas of inhibitory neurons characterized by the decay time constant⁸³. g. Decay time constant of calcium transients of all identified interneurons over days across mice (n = 5 for sound responsive ablation cohort). One-way ANOVA test, p = 0.82. h. Same as Fig. 3f, but change of tuning width by using normalized response amplitudes at 15th largest stimulus index in the tuning curve over days for excitatory (left) and inhibitory (right) neurons. Two-sided t-test of Δnormalized amplitude for excitatory neurons (left) and for inhibitory neurons (right) between baseline days vs. days after ablation with FDR correction for each experimental cohort (* p < 0.05). i. Change in fraction of neuron pairs with high signal correlation and with large response amplitude from baseline among excitatory-excitatory neurons (left), inhibitory-inhibitory neurons (middle), and excitatory-inhibitory neurons (right). Two-sided t-test of Δfraction of excitatory-excitatory neuron pairs with FDR correction in sound responsive cohort on post-ablation days (Sound responsive cohort: p = 0.15, 0.051, 0.11, 0.0034 for day 7, 9, 11, 15). Asterisks on top: † p < 0.055, * p < 0.05, ** p < 0.01. Permutation test for group comparison (asterisk at bottom; * p < 0.05). For inhibitory-inhibitory neuron pairs, sound responsive cohort: p > 0.15 for all post-ablation days; non-sound responsive cohort: p > 0.28 for all post-ablation days; control: p > 0.16 for all post-ablation days. For excitatory-inhibitory neuron pairs, sound responsive cohort: p > 0.25 for all post-ablation days; non-sound responsive cohort: p > 0.30 for all post-ablation days; control: p > 0.26 for all post-ablation days. j. Baseline-normalized best response amplitude of neurons responsive on day 5 in excitatory (left) and inhibitory (right) neurons for the three experimental cohorts. When splitting excitatory and inhibitory neurons based on the responsiveness on day 5, as observed in Fig. 4b, the normalized best response amplitude of neurons responsive on day 5 significantly decreased on day 7 for both the excitatory and inhibitory neurons in the sound responsive ablation cohort, but not in the other cohorts (One-way ANOVA across days for excitatory neurons (left) and for inhibitory neurons (right) in each experimental cohort (asterisks at the right side; ** p < 0.01, *** p < 0.001); Two-sided paired t-test of the normalized best amplitude of excitatory neurons (left: Sound responsive ablation, p = 0.023) and inhibitory neurons (right: Sound responsive ablation, p = 0.049) between day 3 vs. day 7 for each experimental cohort. Asterisks on top; * p < 0.05. k. Same as j, but for newly responsive neurons. For the neurons unresponsive on day 5 but being responsive on the other day, the normalized best response amplitude of excitatory neurons exhibited a delayed but substantial increase after microablation in the sound responsive ablation (One-way ANOVA across days, Sound responsive ablation: F(5, 24) = 7.97, p = 1.5 × 10⁻⁴; asterisk at the right side, * p < 0.01) like Fig. 4b right, but inhibitory neurons did not (p = 0.43). For the non-responsive ablation and the control cohorts, both the excitatory and inhibitory neurons did not change the normalized best response amplitude over days (p ≥ 0.24). Group comparison revealed that the normalized amplitude of the newly responsive excitatory neurons in sound responsive ablation was significantly larger than those in non-responsive ablation and in control late after microablation (Two-sided t-test with FDR correction on a given post ablation day between each two of the three cohorts; two-colored asterisks on top; * p < 0.05). Data presented as mean ± s.e.m. across mice for e, g-k. n = 5 mice for sound responsive ablation cohort, n = 5 mice for non-sound responsive ablation cohort, n = 7 for control for e, h-k. See Supplementary Table 2. for detailed statistics.