A novel discrimination–avoidance task.

A, Top: Schematic of the task. Mice are trained to discriminate between a pair of sounds (lasting 10 s) and shuttle between two distinct rooms of a shuttle box to avoid footshocks. Specifically, sounds A and B signal shocks in rooms A and B, respectively. Bottom: Two behavioral response scenarios. S1: If the mouse makes the correct response, either by staying in or shuttling to the correct room before the sound ends, no shock is administered. S2: If the mouse makes an incorrect response, either by staying in or shuttling to the incorrect room before the sound ends, up to 10 mild shocks are administered until the mouse shuttles to the correct room. Each training session comprises 50 trials, 60 s apart; sounds A and B are presented in a pseudorandom order. B, Learning curve showing the success rate in avoiding shocks across training sessions (mean ± s.e.m.; n = 7). One-way ANOVA: F9, 60 = 8.04; P = 1.07 × 10−7. C, The mean shuttle crossing latencies for correct and incorrect shuttles (averaged over the last two sessions) show a trending significant difference (P = 0.074, paired t test). The black line indicates the mean; grey lines indicate individual mice. Shuttle crossing is defined as the body center crossing the midline opening of the shuttle box.

ACC neurons primarily encode post-action variables.

A, Schematic of an 8-tetrode array and a representative brain section highlighting the recording sites (red arrows) within the ACC. Scale bar, 0.5 mm. B, Diagrams showing a representative shuttle route (left) and corresponding Y-position of the animal’s body center (right). A shuttle response typically lasts ∼1–3 s (Supplementary Fig. 2). C, An example Y-position of the animal’s body center during a stay trial. D, Peri-event rasters and histograms of an ACC neuron during correct-shuttle trials (top; arranged based on shuttle latencies) and correct-stay trials (bottom). E, A representative heatmap activity of simultaneously recorded ACC neurons (n = 29) during correct shuttles (top) and correct stays (bottom) from the same recording session. F, Principal component analysis (PCA) of ACC neuronal population activity as shown in E. PC1, PC2, and PC3 are the first three principal components; the numbers are the percentages of total variance explained by the corresponding PCs. Each circle indicates a time lapse of 0.1 s. Note that there is a robust neural state change in the 3-D PC space surrounding the shuttle response (top), but not the stay response (bottom).

ACC neurons primarily respond during “shuttle” but not “stay” trials.

A&B, Dimension reduction analysis (i.e., PCA) indicates robust changes in ACC neuronal population activity during correct shuttles (A) but not correct stays (B) from four representative recording sessions. Time “0” indicates shuttle crossings (A) or sound onsets (B), respectively. For more details, see Fig. 2.

ACC neurons monitor actions independent of outcomes.

A, Peri-event rasters (trials) and histograms of a representative ACC neuron during correct (left), incorrect (middle), and post-shock shuttles (right) within a session. Red triangles indicate shock administrations. Note that incorrect shuttles are followed by a second shuttle after animals receive footshocks, and approximately half of the post-shock shuttles are preceded by incorrect shuttles. B, Heatmaps showing the activity of individual ACC neurons (n = 348) during correct (left), incorrect (middle), and post-shock shuttles (right). Neurons are arranged in the same order across the three heatmaps. Color bar indicates z-scored activity. Note that the number of incorrect shuttles in a session is often ≤7, leading to greater variability in mean activity. Only sessions with ≥4 incorrect shuttles are included in the analysis. C, Activity indexes of individual ACC neurons between correct and incorrect shuttles (left), and between correct and post-shock shuttles (right). Activity index is defined as: Activity Index = Meanpost-shuttle – Meanpre-shuttle, where Meanpre-shuttle and Meanpost-shuttle are the mean z scores calculated between -5–0 and 0–5 s, respectively, as shown in B. D, Correlation coefficients of the activity (−5–5 s) between correct and incorrect shuttles (x axis) and between correct and post-shock shuttles (y axis). Only the top and bottom quartiles of ACC neurons (as shown in B) are used for the analysis.

ACC neurons monitor action states and action contents.

A&B, Peri-event rasters (trials) & histograms of four simultaneously recorded ACC neurons during two sets of shuttles: rooms A→B shuttles (A) vs. rooms B→A shuttles (B). Note that the first two neurons exhibit indiscriminate responses, either increasing (#1) or decreasing their activity (#2) after the shuttles, thereby monitoring action state changes. In contrast, neurons 3&4 are selectively activated in one set of the shuttles, thereby monitoring action contents (i.e., rooms A→B vs. B→A shuttles). C&D, Z-scored activity of individual ACC neurons (n = 376; recoded from 15 sessions) surrounding rooms A→B shuttles (C) and rooms B→A shuttles (D). Neurons are arranged in the same order in the two heatmaps. E, Principal-component analysis (PCA) classifies ACC neurons into four major categories based on their activity changes surrounding shuttle responses. PC1, PC2, and PC3 represent the first three principal components color coded from low (dark) to high scores (white). Note that most neurons in categories 1 and 3 are action-state neurons, whereas most neurons in categories 2a, 2c, 2e, and 4 are action-content neurons.

Post-shuttle ACC neuronal population activity decodes action content.

C, Schematic diagram of support vector machine (SVM) decoding. ACC neuronal population activity from pre-shuttle period (−5–0), post-shuttle period (0–5), or shuffled spikes is used to train the decoder and subsequently distinguish between action content (rooms A→B vs. B→A shuttles). B, Mean decoding accuracy (blue line) and individual decoding accuracies for 15 sessions (grey lines). One-way ANOVA: F2, 42 = 146.19, P = 1.2 × 10−19. ***P < 0.001, Bonferroni post-hoc test. C, The same as B, except that shorter (left, 2.5 s) or longer decoding windows (right, 7.5 s) were used. ***P < 0.001, paired t test.

Post-action ACC activity influences future performance within a task session.

A, Schematic illustration. B, Mean activity (± s.e.m.) of post-shuttle activated neurons (orange lines; top 1/3), inhibited neurons (blue lines; bottom 1/3), and remaining ACC neurons (black lines; middle 1/3), which preceded either correct trials (solid lines) or incorrect trials (dashed lines). C, Further comparison of the activation strength for post-shuttle activated neurons (left) and inhibited neurons (right) between the two conditions. Each pair of dots indicates an ACC neuron. D–F, Similar to A–C, except that the comparison is based on the status of the preceding trials. Mean z scores in C and F were calculated between 0–5 s after shuttle crossings. Paired t test, n.s., non-significant; *P < 0.05; ***P < 0.001.

Experimental setup.

A, The shuttle box used for behavioral training. Room A is configured with two black walls (left and right), one white wall (back), and a transparent front wall for video recording purposes. Room B is configured with two white walls (left and back), one metal wall (right), and a transparent front wall. B, Schematic diagram of the control setup utilizing MATLAB functions, with numbers indicating the sequence of control flow. C, A comprehensive flowchart illustrating the control setup as shown in B. D, Left, sounds A and B signal shocks in the bottom and top rooms of the shuttle box, respectively. Right, the Y position of a well-trained mouse in a ∼40-min session.

Characterizing ACC neuronal activity in relation to action initiations.

A, Top, schematic illustrating the definition of shuttle initiation, crossing, and termination (pause). Bottom, distributions of half-(left) and full-shuttle durations (right). B, Z-scored activity of all ACC neurons during correct shuttle trials. C, Principal-component analysis (PCA) classifies ACC neuronal activity (as shown in B) into seven categories. PC1, PC2, and PC3 represent the first three principal components color coded from low (dark) to high scores (white). D, Mean activity (± s.e.m.) of the seven categories of ACC neurons. E, Fractions of individual categories of ACC neurons.

ACC shows limited response to cue, stay trials, and footshocks.

Left, Heatmap showing the activity of individual ACC neurons (n = 348) in relation to auditory cue onset. Middle, Heatmap showing the activity of individual ACC neurons (n = 348) during stay trials. C, Heatmap showing the activity of individual ACC neurons (n = 336) during footshock. Note, footshock trials with a shuttle response within 1 s shock onset were excluded to avoid shuttle response confound. As a result, some behavioral sessions were excluded due to insufficient trial numbers.

ACC pyramidal neurons and interneurons both monitor action content.

A, Spike waveforms (mean ± s.d.) of two representative ACC neurons: one putative pyramidal neuron and one interneuron. These two neurons were recorded simultaneously. B, Peri-event rasters (trials) & histograms of the same two ACC neurons surrounding shuttle responses. Both neurons exhibit differential activity changes that discriminate between rooms A→B (top panels) vs. B→A shuttles (bottom panels).

ACC neurons do not display place cell activity characteristics.

A, Left, a representative path throughout all inter-trial intervals during a training session, excluding data within 5 s of any shuttle responses. Middle and right, place field activity of two representative ACC action-content neurons. Both neurons show spiking activity across the chamber without place preference. The color bar indicates spikes/s. B, Similar to A but from a different animal. Note that these neurons are the same neurons depicted in Figure 5 (#3 & #4).

A simplified model of information integration during complex cue–action– outcome associative learning.

The integration occurs around the time of outcome delivery, when all three sets of information (cue, action, and outcome) are available.