Experimental design, behavioral responses, viral strategy, and calcium imaging.

a. Experimental design illustrating discriminative fear conditioning with either a 15-kHz CS+ or a 3-kHz CS+ (conditioned groups) and no-shock controls, which were tested at the same time points but never received footshocks. All groups were tested with 3- and 15-kHz tones and two intermediate frequencies (7 and 11 kHz) on days 1, 15, and 30 after conditioning. b. Behavioral responses across retrieval sessions (CS+15 group: n = 27 mice; CS+3 group: n = 22 mice; no-shock controls: n = 13 on days 1 and 15 and n = 11 on day 30). Two control mice lost their lenses after day 15 and therefore did not contribute data to the day 30 session. The repeated-measures analysis included the 11 control mice with complete data across all sessions. Two-way repeated-measures ANOVA. CS+15 group: main effect of day, F(2,52) = 13.28, p < 0.001; main effect of frequency, F(3,78) = 85.09, p < 0.001; and day × frequency interaction, F(6,156) = 3.79, p = 0.002. CS+3 group: main effect of day, F(2,42) = 14.42, p < 0.001; main effect of frequency, F(3,63) = 58.81, p < 0.001; and day × frequency interaction, F(6,126) = 1.41, p = 0.217. No-shock controls: main effect of day, F(2,20) = 1.38, p = 0.276; main effect of frequency, F(3,30) = 1.43, p = 0.253; and day × frequency interaction, F(6,60) = 0.335, p = 0.916. Significant pairwise comparisons following Tukey’s multiple-comparisons test are denoted by asterisks: ***p < 0.001. c. Viral strategy and representative histological section showing GCaMP6f expression. Imaging depth did not exceed 300 μm, restricting recordings to the PL. d. Representative calcium-imaging data showing a raw fluorescence frame, processed frame, maximum-intensity projection across frames, and corresponding activity traces.

Distribution of stable and dynamic cell types across sessions.

a. Activity plots from a representative animal conditioned with a 15-kHz CS+, with neurons ordered by activity level. The plots show positive tone-responsive neurons (bottom), negative tone-responsive neurons (top), and mixed or nonresponsive neurons (middle) before and after tone presentation. b. Proportions of neuronal response types across experimental groups and sessions. Numbers of cells—CS+15 group: conditioning, 3,834; day 1, 3,177; day 15, 4,186; and day 30, 3,693. CS+3 group: conditioning, 1,628; day 1, 1,381; day 15, 1,881; and day 30, 1,833. No-shock controls: conditioning, 1,118; day 1, 986; day 15, 1,386; and day 30, 563. Two control mice yielded data only through day 15. c. Venn diagrams showing the proportions of consistently active neurons (active during all three retrieval sessions), partially active neurons (active during two sessions), and transiently active neurons (active during only one session). The proportions of neurons in these categories were similar across groups, except that transient neurons on day 30 were more abundant in the control group. This difference reached significance only between the control and CS+15 groups (p < 0.05; Table S1). Diagrams and analyses for the control group included only mice recorded through day 30.

Population activity of positive tone-responsive neurons exhibits graded patterns reflecting learned threat value.

a–c. Population responses in animals conditioned with a 15-kHz CS+ (a), animals conditioned with a 3-kHz CS+ (b), and no-shock controls (c). In each group, the upper panels show positive tone-responsive neurons, and the lower panels show negative tone-responsive neurons. Adjacent boxplots show the areas under the population response curves (AUCs). Boxplots indicate the median (center line) and interquartile range (box); whiskers extend to the most extreme values within 1.5 times the interquartile range, and points beyond the whiskers represent individual outliers. CS+15 group—positive responders: day 1, F(3,18) = 9.963, p < 0.001; day 15, F(3,18) = 9.973, p < 0.001; and day 30, F(3,18) = 6.627, p = 0.003. Negative responders: day 1, F(3,18) = 2.483, p = 0.094; day 15, F(3,18) = 1.877, p = 0.178; and day 30, F(3,18) = 2.753, p = 0.073. CS+3 group—positive responders: day 1, F(3,12) = 6.899, p = 0.006; day 15, F(3,12) = 9.247, p = 0.002; and day 30, F(3,12) = 6.123, p = 0.009. Negative responders: day 1, F(3,12) = 0.870, p = 0.484; day 15, F(3,12) = 0.512, p = 0.641; and day 30, F(3,12) = 1.448, p = 0.278. No-shock controls—positive responders: day 1, F(3,15) = 0.527, p = 0.670; day 15, F(3,15) = 1.852, p = 0.181; and day 30, F(3,9) = 1.046, p = 0.418. Negative responders: day 1, F(3,13) = 1.205, p = 0.347; day 15, F(3,13) = 1.375, p = 0.294; and day 30, F(3,9) = 0.950, p = 0.457. Asterisks denote significant Tukey post hoc comparisons: *p < 0.05, **p < 0.01, and ***p < 0.001.

Generalized linear model (GLM) analysis of the relative contributions of tone identity and freezing behavior to neuronal activity for positive (a-d) and negative (e-h) responder cells.

a. Regression coefficients (β) for positive tone-selective neurons in conditioned mice exhibit opposite monotonic gradients across tones after controlling for freezing. This graded pattern is absent in no-shock controls. b. The freezing-to-tone coefficient ratio remains below 1 for every tone in all groups, indicating a greater contribution of tone identity than freezing to the activity of positive tone-selective neurons. c. Within-cell comparisons confirm that the absolute median β coefficients for tone exceed the absolute freezing coefficients in all groups. d. When pooled across tones, the proportion of freezing-dominant neurons remains stable across retrieval sessions. e. Negative tone-selective neurons exhibit suppressive tone coefficients across all frequencies, with only weak frequency dependence, indicating the absence of pronounced graded responses. f. Freezing-to-tone coefficient ratios are higher than those observed in positive responders but remain below 1 across tones in conditioned mice. In no-shock controls, the ratio approaches 1, indicating similar contributions of freezing and tone identity. g. Within-cell comparisons confirm that the absolute tone coefficients exceed the absolute freezing coefficients, but only in the conditioned groups. h. The proportion of freezing-dominant neurons remains relatively stable across retrieval sessions in each group. Controls displayed a slightly higher proportion of freezing-dominant neurons, but this difference was not significant. Significant Tukey multiple comparisons are denoted by asterisks: *p < 0.05, **p < 0.01, and ***p < 0.001.

Population-vector similarity across tones and time.

a–c. Population-similarity maps for all tone pairs across the time course of tone presentation in the CS+15 (a), CS+3 (b), and no-shock control (c) groups. d. Schematic illustrating the color scale and orientation of the similarity maps. The y-axis represents the earlier tone in each comparison, and the x-axis represents the later tone. e, f. Boxplots showing population similarity during the first 5 s after tone onset, quantified relative to the CS+, for the CS+15 (e; F(3,36) = 12.025, p < 0.001) and CS+3 (f; F(3,24) = 7.435, p = 0.004) groups. Asterisks denote significant Tukey post hoc comparisons: *p < 0.05, **p < 0.01, and ***p < 0.001.

Signed mutual information (MI) clustering identifies PL subpopulations with distinct tone-response profiles.

a. MI-based clustering pipeline. An MI matrix generated from simultaneously recorded PL neurons was spectrally clustered according to shared information, independently of response sign. Within each primary cluster, MI values were combined with the signs of pairwise correlations and reclustered to identify subclusters with distinct positive or negative activity patterns. Subcluster labels were then combined to generate the final signed MI matrix. b. Clustered signed MI matrices for the CS+15 (top), CS+3 (middle), and no-shock control (bottom) groups, with neurons sorted by cluster label. Color indicates the sign and magnitude of MI. c. Mean stimulus-aligned responses of clusters positively modulated by individual tones. c.1–c.4 correspond to 3, 7, 11, and 15 kHz, respectively. d. Mean stimulus-aligned responses of clusters negatively modulated by individual tones. d.1–d.4 correspond to 3, 7, 11, and 15 kHz, respectively.

Graded tone-responsive neurons are present only in conditioned groups.

a, b. Mean stimulus-aligned population responses of clusters exhibiting graded tone responses in animals conditioned with a 15-kHz CS+, showing positive (a) and negative (b) response patterns. c–e. Mean stimulus-aligned population responses of clusters exhibiting graded tone responses in animals conditioned with a 3-kHz CS+, showing positive (c, d) and negative (e) response patterns. f, g. Stability of neuronal identity within graded tone-responsive clusters across retrieval sessions. Asterisks denote significance after Benjamini–Hochberg correction. h, i. Baseline-to-stimulus firing-rate ratios (BSRs) showing changes in the activity of graded clusters across retrieval sessions in the CS+15 (h) and CS+3 (i) groups. Graded clusters were not identified in no-shock controls. Asterisks denote significant Tukey post hoc comparisons: *p < 0.05, **p < 0.01, and ***p < 0.001.

GLM analysis of the contributions of tone identity and freezing to the activity of clustered PL neurons.

a–h. Positive (a–d) and negative (e–h) single-tone-selective neurons. a. Tone β coefficients in conditioned mice form opposing gradients that peak at the CS+; these gradients are absent in no-shock controls. b. Freezing-to-tone ratios remain below 1 across preferred tones and retrieval sessions. c. Within-cell comparisons confirm that absolute tone coefficients exceed absolute freezing coefficients in all groups. d. The proportion of freezing-dominant neurons is low and stable across retrieval sessions. e. Negative tone-selective neurons exhibit suppressive tone β coefficients with weak frequency dependence and greater variability in no-shock controls. f. Freezing-to-tone ratios are higher than those of positive responders but remain below 1 in conditioned mice. g. Within-cell comparisons show that absolute tone coefficients exceed absolute freezing coefficients in negative responders from conditioned mice, although by a smaller margin than in positive responders. h. The proportion of freezing-dominant neurons is higher than among positive responders but remains stable across retrieval sessions. i–p. Positive (i–l) and negative (m–p) graded neurons. i. Tone β coefficients obtained after controlling for freezing exhibit strong CS+-centered gradients. j. Freezing-to-tone ratios are well below 1, indicating that tone identity contributes substantially more strongly to neuronal activity than freezing. k. Within-cell comparisons confirm that absolute tone coefficients greatly exceed absolute freezing coefficients. l. Only a small and stable proportion of positive graded neurons is freezing-dominant. m. Negative graded neurons exhibit strong suppressive gradients. n. Freezing-to-tone ratios remain below 1. o. Absolute tone coefficients are approximately threefold greater than absolute freezing coefficients. p. The proportion of freezing-dominant neurons remains low across retrieval sessions. Asterisks denote significant Tukey post hoc comparisons: *p < 0.05, **p < 0.01, and ***p < 0.001.