The mPFC→NAc and mPFC→BLA neurons demonstrated distinct activity patterns across various emotional states.

(A) Schematic showing retrograde AAV-GCaMP6 injection in the NAc and BLA, respectively. In vivo Ca2+ imaging was performed in the mPFC neurons expressing GCaMP6 via miniscope.

(B) Top: An example of AAVretro-GCaMP6 injection and expression in the BLA and NAc, respectively. Bottom: An example of GCaMP6m expression in the mPFC with AAVretro- GCaMP6 injection in the NAc and an image of Ca2+ fluorescence recorded with the miniaturized microscope. Green, GCaMP6m. Scale bars, 200 mm.

(C) Left: the field of view under a GRIN lens in one mouse with identified neurons numbered and colored. Right: fluorescence traces of example neurons marked in the above panel.

(D) Schematic of the open field test (OFT) in an open 50 cm X 50 cm arena. The green areas represent corners and the orange area represents the center.

(E) Illustrations depicting typical mouse behaviors associated with different emotional states observed during the OFT: Center (exploration and higher anxiety), Corner (lower anxiety), sniffing (sensory exploration and vigilance, possibly indicating curiosity, cautious exploration, or heightened alertness), and grooming (stress relief or comfort).

(F) The percentage of time that mice spent in the center, corners, sniffing, and grooming during a 10-min open field test. n = 15 mice for mPFC→BLA group; n = 11 mice for mPFC→NAc group. The behavioral performance between these two groups of mice showed no significant difference.

(G) Heatmap and time-series plot of neuronal activity (ΔF/F) across the test duration. The heatmap above shows the fluctuation in activity level across neurons, while the plot below aligns the averaged fluctuations of these neurons with observed behaviors (Center, Corner, Sniffing, Grooming) within the arena.

(H) Normalized transient rates of neuronal activity during different behaviors. Both mPFC→BLA and mPFC→NAc pathways are plotted, indicating no significant variation in neuronal firing rates across behaviors.

(I) PCA plots for mPFC→BLA and mPFC→NAc pathways illustrating the distribution of neuronal activity patterns for different observed behaviors (Center: blue, Corner: red, Sniffing: green, Grooming: purple). Each point represents an individual neuronal recording.

(J) Summary of the distances of neuronal activity clusters from the center of Corner behavior in the OFT for both pathways

Data are represented as mean ± SEM. * p < 0.05, *** p < 0.001, Mann-Whitney U test.

Distinct encoding of emotional status by center-ON neurons in the mPFC pathways

(A) Ca2+ traces of the averaged activity of center-ON neuron ensembles around the onset of center entry (5 s before to 5 s after). Solid lines represent the averaged value and shaded regions indicate SEM.

(B) Heatmaps depicting the activity patterns of neurons across four different behavioral contexts: Center, Corner, Sniffing, and Grooming. Each column corresponds to a different behavior, with the intensity of color indicating the level of neuronal activity.

(C) Left: spatial distributions of center-ON neurons among the mPFC→BLA and mPFC→NAc neurons in one representative mouse from each group. Right: the percentage of center-ON neurons within the mPFC→BLA and mPFC→NAc neuron ensembles.

(D) Spatial heatmaps of neuronal activity across the open field arena of all observed center-ON neurons in one example mouse of mPFC→BLA and mPFC→NAc groups. The color bar indicates the averaged normalized z-score.

(E) Averaged transient rate in four different states during the OFT.

(F) PCA plots illustrating the distribution of neuronal activity patterns during different behaviors for mPFC→BLA and mPFC→NAc pathways. Points are color-coded by behavior type (Center: blue, Corner: red, Sniffing: green, Grooming: purple).

(G) Summary of the distances from the center of Corner behavior in terms of neuronal activity for each behavior in both mPFC pathways.

Data are represented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, Mann-Whitney U test.

The mPFC→NAc and mPFC→BLA neurons demonstrated distinct activity patterns across various emotional states during the EPM test.

(A) Schematic illustration of the Elevated Plus Maze (EPM) test setup showing the layout of open (Open-P, Open-NP) and closed (Close-P, Close-NP) arms used to assess anxiety-related behaviors in mice.

(B) Box plots displaying the percentage of time spent by mice in the different arms of the EPM (Open-P, Open-NP, Close-P, Close-NP) for both mPFC→BLA and mPFC→NAc pathways. Data indicate variations in time spent across different arms, highlighting behavioral preferences.

(C) Averaged transient rates (Hz) of neuronal activity of all recorded neurons in the mPFC→BLA and mPFC→NAc pathways in each arm of the EPM. Neuronal activity patterns are compared across these two pathways, showing no significant difference.

(D) Analysis design of observing the neural activities of center-ON ensembles of OFT in different EPM arms.

(E) Mean Ca2+ signal across the EPM arms of all observed center-ON neurons in one example mouse. The color bar indicates the averaged normalized z-score.

(F) Example 30 center-ON neurons (top) and corresponding averaged activity (bottom) around the onset of arm entry during the EPM test. Time 0 represents the onset of arm entry.

(G) Averaged transient rate in four different arms during the EPM test. n = 9-13 mice mPFC→BLA group; n = 5-8 mice for mPFC→NAc group.

Data are represented as mean ± SEM. * p < 0.05, Mann-Whitney U test.

Pattern decorrelation is shown by the mPFC→NAc neurons, but not mPFC→BLA neurons.

(A) Top: the apparatus for the social interaction test. The subject mouse is in the middle 45 cm- long chamber; the 10 cm end compartments contain different stimuli. Bottom: the three sessions of the social interaction test. In the first 10-minute test session (S1), a strange mouse (M1) and an object (O) were placed in the end chambers; session 2 (S2) used the same stimuli but swapped their positions; in session 3 (S3) a new mouse (M2) replaced O, so that the subject mouse must choose whether to interact with a familiar or strange mouse (M1 vs. M2).

(B) The percentage of time that mice (n = 10) spent interacting with stimuli in each session.

(C-D) The averaged Ca2+ event rate when mice were engaged with different stimuli over the three sessions. n = 14 mice mPFC→BLA group; n = 11 for mPFC→NAc group. Data are represented as mean ± SEM. The event rates during interaction with M1 and O did not show statistical significance across all three sessions for both the mPFC→BLA and mPFC→NAc groups, two-way RM-ANOVA with Bonferroni–corrected post hoc comparisons.

(E-F) Top: raster plots of correlation coefficient of paired mPFC neurons during interactions with different stimuli, from a representative mouse in mPFC→BLA and mPFC→NAc group, respectively. Bottom: distribution of pair-wise Pearson correlation coefficients among these recorded mPFC neurons in responding to stimuli in each session. The blue and orange plots represent M1 interactions, while the lighter colors represent the interaction with the other stimulus (O).

(G) The averaged full width at half maximum (FWHM) of correlation coefficient distribution of individual mice in mPFC→BLA (n = 11) and mPFC→NAc (n = 10) group during interactions with different stimuli.

Data are represented as mean ± SEM. * p < 0.05, *** p < 0.001, Mann-Whitney U test.

Comparative analysis of all neurons and center-ON subsets reflects divergent encoding patterns in the mPFC→BLA and mPFC→NAc Pathways.

(A) PCA plots showing the activity of all recorded neurons in the mPFC→BLA and mPFC→NAc pathways during interactions with a social stimulus (mouse, M1) and a nonsocial stimulus (object, O) in a modified Three Chamber Test (mTC). The plots reveal distinct clustering of neural activity patterns for each stimulus within both pathways.

(B) Distances between all neuronal activity clusters for nonsocial (O) interactions from the center of that for M1 interaction, compared across mPFC→BLA and mPFC→NAc pathways. Significant difference is demonstrated in the mPFC→BLA pathway.

(C) PCA plots for center-ON neurons identified during the Open Field Test (OFT). These plots illustrate the activity of these neurons in the mPFC→BLA and mPFC→NAc pathways during the same social and nonsocial stimuli. Clusters show how Center-ON neurons specifically respond to each type of stimulus.

(D) Distances of all center-ON neuronal activity clusters from the center of that for M1 interaction, compared across mPFC→BLA and mPFC→NAc pathways. Significant difference is demonstrated in the mPFC→NAc pathway.

Data are represented as mean ± SEM. *** p < 0.001, Mann-Whitney U test.

The modifications in the social ranking of mice alter their anxiety and social states.

(A) Schematic illustrating the experimental scheme. Mice underwent an open field test, followed by five consecutive days of tube tests (once daily) to establish their social rankings. Subsequently, both the winner and loser mice underwent a repeat open field test to assess their anxiety states.

(B-C) The mice’s total distance traveled in the open field and time spent in the center zones before and after the tube test. * p< 0.05; ns, non-significant. n = 6 mice for each group, winner vs loser, unpaired Student’s t-test.

(D) The differences in total distance (top) and center time (below) between winner and loser groups before and after the tube tests. *** p < 0.001; ns, non-significant. Before vs. after tube test, Two-way ANOVA Multiple comparisons, Sidak’s post-hoc test.

(E) Schematic illustrating the three-chamber test for social ability and social memory. Mice were introduced to the middle chamber, which comprised two smaller side chambers, and underwent three 10-minute sessions. In Session 1, mice acclimated to the apparatus without the presence of another mouse or object (O). For the social ability test (session 2), a novel mouse (M1) occupied one side chamber, while an object was placed in the other. In the social memory test (session 3), the previously encountered mouse (M1) remained in one side chamber, while a new mouse (M2) replaced the object in the other side chamber. After the three-chamber test, the mice underwent tube tests to establish their social rankings followed by a repeat three-chamber test to assess their social states.

(F) The mice’s exploration time near M1 and O/M2 during the social ability test (left) and social memory test (right) conducted before the tube test. Social ability index calculated as (time near M1 chamber - time near object chamber) / (time near M1 chamber + time near object chamber). The social memory index is calculated as (time near M2 chamber - time near M1 chamber) / (time near M2 chamber - time near M1 chamber). * p < 0.05, *** p < 0.001, **** p < 0.0001, ns, non-significant. Two-way ANOVA Multiple comparisons, Sidak’s post- hoc test. Unpaired Student’s t-test for preference index.

(G) The mice’s exploration time was spent near the M1 and O/M2 during the social ability test and social memory test after the tube test. * p < 0.05, *** p < 0.001, **** p < 0.0001. Two- way ANOVA Multiple comparisons, Sidak’s post-hoc test. Unpaired Student’s t-test for preference index.

(H) The comparisons of the social ability index (top) and social memory index (below) of the winner and loser group before and after the tube tests. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, non-significant. Two-way ANOVA Multiple comparisons, Sidak’s post-hoc test). n = 6 mice for each group.

The social status-dependent PL-NAc and PL-BLA neuronal activities.

(A) Schematic showing the AAV-ChR2 injection in the PL of mPFC.

(B) Expression of ChR2-EYFP in the mPFC, and terminal expression of ChR2-EYFP in the NAc and BLA. Scale bar, 400 μm, 50 μm, and 50 μm, from left to right.

(C) The ChR2-EYFP expressed mice underwent the tube test to determine their winner and loser status.

(D) The EPSCs in the NAc were evoked by 470 nM blue light. Upper, schematic showing the light fiber placement and EPSC recording sites in the NAc; Lower, representative EPSCs in the brain slices from the winner (red) and loser (black) mice. Scale bar, 30 pA and 25 ms.

(E) The normalized light intensity-dependent EPSCs. The data points were normalized to 10 μW and were shown as mean values. Winner, n= 19 neurons/ 7 mice; Loser n= 14 neurons/ 7 mice.

(F) The representative pair pulse ratio (PPR) of the EPSCs in the NAc (the light intensity was 1- 2 μW and the interpulse interval was 50 and 75 ms). Scale bar, 8 pA and 25 ms.

(G) Summarized plots of the PPR under different interpulse intervals. Winner, n= 7 neurons/ 7 mice; Loser, n = 14 neurons/ 7 mice. ns, non-significant. Scale bar, 8 pA and 25 ms.

(H-I) EPSC recordings in the BLA. Winner, n = 17 neurons/ 7 mice; Loser n = 21 neurons/ 7 mice. Scale bar, 6 pA and 25 ms.

(J) The representative PPR of the EPSCs in the BLA.

(K) Summarized plots of the PPR under different interpulse intervals. Winner, n = 8 neurons/ 7 mice; Loser, n = 11 neurons/ 7 mice. Scale bar, 3 pA, and 25 ms.

(L) Left, representative EPSCs in the PL-NAc neurons from winner (red) and loser (black) mice. Right, the summarized AMAP/NMDA ratio. Winner, n= 5 neurons/ 5 mice; Loser n= 7 neurons/ 7 mice, 1-5 ms pulse). Scale bar, 25 pA and 50 ms.

(M) Left, representative EPSCs in the PL-BLA neurons from winner (red) and loser (black) mice. Right, the summarized AMAP/NMDA ratio. Winner, n= 12 neurons/ 7 mice; Loser n= 21 neurons/ 7 mice, 1-10 ms pulse). Scale bar, 10 pA, and 50 ms.

Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. Two-way ANOVA for C, G, I, and K; two-tailed paired Student’s t-test for L and M.

ChR2 currents in the PL neurons from the winner and loser mice.

(A) Schematic showing the light fiber placement and EPSC recording sites in the mPFC.

(B) The representative blue light intensity-dependent photocurrents.

(C) The normalized light intensity-dependent photocurrents in the neurons from winner (red) and loser (black) mice. Winner, n= 12 neurons/ 7 mice; Loser n= 16 neurons/ 7 mice.

(D) The representative traces of action potentials induced by blue lights.

(E) The summarized numbers of action potentials and the frequency of light stimulations. Winner, n= 7 neurons/ 4 mice; Loser n= 7 neurons/ 4 mice.

Data are shown as mean ± SEM. Two-way ANOVA. No significant difference was observed between the winner and loser groups.