Histological verification of lens locations in the mPFC and immunohistochemical and morphological characterization of GCaMP6f-expressing neurons.

(a) Histological verification of recording sites (1 mm lenses) in the prelimbic (PrL) and cingulate cortex (Cg1). M2: secondary motor cortex, IL: infralimbic cortex, DP: dorsal peduncular cortex. Yellow lines: estimated imaging planes. (b) Percentage of neurons recorded in different brain regions. (c) Zoomed-in view of a z-projected confocal image stack below the imaging lens. (d) GCamP6f-positive neurons display spiny dendrites. Inset: Enlarged views with spines indicated by arrows. (e) Confocal z-projection of GCamP6f-positive neurons (left) and reconstructed apical dendrites from the same field of view (right) showing characteristic dendritic morphologies of pyramidal neurons. (f) Measurement of the colocalization of GCaMP6f with markers of pyramidal cells (Emx1, n = 1 mouse) and GABAergic interneurons (parvalbumin (PV) and somatostatin (SOM), n = 3 mice). Examples of immunostainings (left) and neurons that were positive for GCaMP6f and/or one of the marker proteins (right). Positive neurons were detected with the CellPose algorithm [44]. (g) Top: Quantification of the percentage of marker-positive cells that coexpress GCaMP6f (n = 1,869 Emx1-positive neurons, n = 412 PV-positive neurons, n = 377 SOM-positive neurons tested). A large fraction of Emx1-positive pyramidal cells coexpressed GCaMP6f while expression was rare in interneurons. Bottom: Percentage of GCamP6f-positive neurons expressing Emx1, PV or SOM (n = 708, 2,412, and 2,787 GCaMP6f-positive cells tested). The vast majority of GCaMP6f-positive neurons expressed Emx1 while expression of interneuron markers was rare. Dots show mouse averages. The data underlying this figure can be found at https://doi.org/10.5281/zenodo.10378756. (EPS)</p

Illustration of the manifold alignment procedure.

Intense threat elicits action in the form of active and passive coping. The medial prefrontal cortex (mPFC) executes top-level control over the selection of threat coping strategies, but the dynamics of mPFC activity upon continuing threat encounters remain unexplored. Here, we used 1-photon calcium imaging in mice to probe the activity of prefrontal pyramidal cells during repeated exposure to intense threat in a tail suspension (TS) paradigm. A subset of prefrontal neurons displayed selective activation during TS, which was stably maintained over days. During threat, neurons showed specific tuning to active or passive coping. These responses were unrelated to general motion tuning and persisted over days. Moreover, the neural manifold traversed by low-dimensional population activity remained stable over subsequent days of TS exposure and was preserved across individuals. These data thus reveal a specific, temporally, and interindividually conserved repertoire of prefrontal tuning to behavioral responses under threat.</div

Rate coding of passive and active coping responses.

(a) Decoding of TS struggle/immobility from calcium data. Left: Simultaneously recorded neurons (n = 301) in one mouse during the first day of TS exposure (sorted by their correlation to TS movement) along with movement speed (top). Right: Results of decoding with real vs. surrogate data created by randomly shifting calcium traces in time (t = 11.26, p = 9 * 10−5, paired t test, n = 6 mice). (b) Correlation of d1 movement scores during TS and baseline (i.e., standardized β-coefficient between the animal’s speed and each neuron’s calcium trace). Distributions of baseline and TS movement scores are shown on the top and side, respectively. Solid lines show the median, dashed lines the first and third quartiles. β = −0.04 ± 0.05, p = 0.423, LME. Right: Stronger movement tuning during TS as quantified from the average absolute movement score of each mouse. t = −15.0, p = 10−5, paired t test, n = 6 mice. (c) Linear model predicting calcium signals of individual neurons with movement. Left: Summary of explained variance (EV) during baseline (n = 277 cells) and TS (n = 160 cells, U = 9,313, p = 10−24, Mann–Whitney U test). Right: Examples of true (black) and predicted calcium signals (colored) during both states. (d) Proportion of neurons for which calcium activity could be significantly explained by movement alone (t = −3.82, p = 0.012, paired t test). (e) Decoding baseline and TS motion speed with models trained on TS (top) or baseline (bottom) population calcium data. Examples of one mouse are shown with the true speed in black and the predicted speed in color. Pearson’s correlation coefficient for each fit is shown on top. (f) Quantification of the data shown in (e) for all mice. Trained on TS: TS vs. baseline: t = 12.43, p = 0.0004, TS vs. shuffle: t = 13.77, p = 0.0003, baseline vs. shuffle: t = 2.51, p = 0.375. Trained on baseline: baseline vs: TS: t = 3.89, p = 0.08, baseline vs. shuffle: t = 8.28, p = 0.003, TS vs. shuffle: t = 0.26, p = 1.0. Trained on TS predicting TS vs. trained on baseline predicting baseline; t = 8.09, p = 0.0033, one-way repeated measures ANOVA followed by paired t tests with Bonferroni correction. Dashed lines: chance level. n = 6 mice, *p https://doi.org/10.5281/zenodo.10378756.</p

Across-day predictions with manifolds constructed with other dimensionality reduction methods and from single day-active neurons.

(a) Example manifold alignments using principal component analysis (PCA, left) and spectral embedding (right). (b) Aligned manifolds based on PCA predict TS struggle/immobility (d3: vs. reference: z = 0, p = 1, vs. random: t = 16.2, p = 5 * 10−5, vs. shuffled: t = 14.09, p = 9 * 10−5, d9: vs. reference: t = 1.12, p = 0.787, vs. random: t = 16.94, p = 4 * 10−5, vs. shuffle: t = 17.41, p = 3 * 10−5) and change in speed with comparable precision to the within-day reference (d3: vs. reference: t = 1.51, p = 0.571, vs. random: t = 39.07, p = 6 * 10−7, vs. shuffled: t = 10.51, p = 0.0005, d9: vs. reference: t = 1.52, p = 0.564, vs. random: t = 98.31, p = 6 * 10−9, vs. shuffle: t = 21.26, p = 10−5). (c) Same as (b) but for spectral embedding used to create manifolds (struggle/immobility prediction: d3: vs. reference: t = 0.49, p = 1, vs. random: t = 19.1, p = 2 * 10−5, vs. shuffled: t = 17.71, p = 3 * 10−5, d9: vs. reference: t = 0.49, p = 1, vs. random: t = 27.63, p = 3 * 10−6, vs. shuffle: t = 10.85, p = 4 * 10−4; change in speed: d3: vs. reference: t = 0.89, p = 1, vs. random: t = 24.23, p = 6 * 10−6 vs. shuffled: t = 20.07, p = 2 * 10−5, d9: vs. reference: t = 1.03, p = 1, vs. random: z = 2.88, p = 0.012, vs. shuffle: t = 12.45, p = 2 * 10−4). (d) Performance of Isomap embedding is robust against the number of dimensions used when predicting struggle/immobility (p = 0.694) and change in speed (p = 0.51). (e) Frame-by-frame prediction of TS struggling/immobility predicted from aligned manifolds of neurons found active only on a single recording day trained on d1 (left, d3: vs. reference: t = 0.69, p = 1, vs. random: t = 12.28, p = 0.0002, vs. shuffled: t = 6, p = 0.006, d9: vs. reference: t = 0.69, p = 1, vs. random: t = 10.77, p = 3 * 10−4, vs. shuffle: t = 11.62, p = 3 * 10−4). (f) Same as (e) but predicting change in speed (d3: vs. reference: t = 2.17, p = 0.247, vs. random: t = 20.29, p = 2 * 10−5, vs. shuffle: t = 11.17, p = 3 * 10−4, d9: vs. reference: t = 2.01, p = 0.302, vs. random: t = 37.54, p = 7 * 10−7, vs. shuffle: t = 17.62, p = 3 * 10−5). Dashed lines: chance level. One-way repeated measures ANOVAs followed by paired t tests or Wilcoxon rank sum tests with Bonferroni correction, n = 6 mice. *p https://doi.org/10.5281/zenodo.10378756. (EPS)</p

Additional analyses of TS selectivity and prediction of baseline and TS state with different decoding models.

(a) TS selectivity scores (as shown in Fig 1e) plotted separately for prelimbic (PrL, n = 704 neurons) and cingulate (Cg, n = 126 neurons) areas. t = 2.96, p = 0.003, unpaired t test. (b) Neurons of both brain regions show comparable correlation in their TS selectivity on days 1 and 9 (PrL: Pearson’s r = 0.68, p = 3 * 10−97, Cg: r = 0.60, p = 10−13). (c) TS selectivity scores plotted separately for female (n = 275 neurons from 2 mice) and male mice (n = 552 neurons from 4 mice). There was no difference between both sexes (t = 0.61, p = 0.544, unpaired t test). (d) Neurons of both sexes show comparable correlation in their TS selectivity on days 1 and 9 (females: Pearson’s r = 0.69, p = 10−40, males: r = 0.69, p = 4 * 10−80). (e) Prediction of baseline vs. TS state with different decoding models. Left: Using a support vector machine, accuracy across days was lower compared to within-d1 (d3: t = 4.93, p = 0.021, d9: t = 6.36, p = 0.007) but above chance level (vs. shuffled: d1: t = 224.22, p = 10−10, d3: t = 31.41, p = 10−6, d9: t = 33.36, p = 10−6). Middle: Same comparison between days (d3: t = 3.1, p = 0.134, d9: t = 3.49, p = 0.087) and shuffle (d1: t = 43.83, p = 10−7, d3: t = 36.84, p = 10−6, d9: t = 25.36, p = 10−6) using Gaussian naive bayes classifier. Right: Across days (d3: t = 4.7, p = 0.027, d9: t = 9.54, p = 0.001) and shuffle (vs. shuffled: d1: t = 107.74, p = 10−9, d3: t = 27.74, p = 10−6, d9: t = 49.49, p = 10−7) comparisons using a ridge classifier. One-way repeated measures ANOVAs followed by paired t tests with Bonferroni correction, n = 6 mice. *p https://doi.org/10.5281/zenodo.10378756. (EPS)</p

Examples and metrics of cell registration across days.

(a) Example field-of-view of all neurons detected on each individual day and their overlap with neurons detected on all other days. Inset: Quantification of true positive (left) and true negative cell detection scores obtained with the CellReg algorithm [47]. Data points are mouse averages. (b) Example of stable spatial locations in the field of view and stable response properties. The ROIs shown are classified as struggle-selective neurons (see Fig 3). A short segment of calcium activity during TS is shown for 2 of the cells. Both neurons maintain their struggle-related activity over days. The data underlying this figure can be found at https://doi.org/10.5281/zenodo.10378756. (EPS)</p

Manifold structure of individual mice.

Left graphs: 2-dimensional trajectories of all struggle and immobility epochs during d1 of TS, color coded by relative start and end times. Middle graphs: Flow fields overlaid on the same figures. Right graphs: Angles between consecutive velocity vectors on the same 2-dimensional space illustrating that population activity follows a consistent rotational trajectory in each animal. The data underlying this figure can be found at https://doi.org/10.5281/zenodo.10378756. (EPS)</p

Temporal evolution of the neural manifold during TS.

Projection of the manifold obtained from nonlinear dimensionality reduction. Struggling is shown in blue, immobility in red, with hue value indicating the relative progression through each individual epoch of struggling/immobility. Video shows 2× speed. (MP4)</p

Manifold structure during TS is preserved across individuals.

(a) Examples of manifolds of mice aligned to the orientation of the manifold from another individual. (b) Speed during TS predicted with aligned manifolds trained on the reference subject. Same pairs of mice as shown in (a). (c) Predictions of TS struggling/immobility (vs. reference (i.e., within mice): t = 0.24, p = 1, vs. random: t = 25.83, p = 4 * 10−6, vs shuffle: t = 7.4 p = 0.002) and correlation between predicted and true speed between mice (vs. reference: t = 0.44, p = 1.0, vs. random: t = 27.96, p = 3 * 10−6, vs. shuffle: t = 16.31, p = 4 * 10−5). Dashed lines: chance level. One-way repeated measures ANOVAs followed by paired t tests with Bonferroni correction, n = 6 pairs of mice. ***p https://doi.org/10.5281/zenodo.10378756.</p

Stable population coding of threat coping responses over time.

(a) Examples of an immobility-active (top) and struggling-active neuron (bottom) over recording days 1, 3, and 9. (b) Top: Correlation of TS movement scores on d1 and d3 (top) and mouse averages of correlations to d1, β = 0.60 ± 0.04, p t test. (c) Decoding TS behavior on subsequent days using models trained on calcium activity of repeatedly active neurons on d1. Top: example of predicted speed on d3. Bottom: Prediction of struggling/immobility (left, d3: vs. d1: t = 2.07, p = 0.374, vs. shuffle: t = 13.91, p = 10−4, d9: vs. d1: z = 1.92, p = 0.219, vs. shuffle: z = 2.88, p = 0.016) and correlation of speed (d3: vs. d1: t = 0.52, p = 1, vs. shuffle: t = 19.84, p = 2 * 10−5, d9: vs. d1: t = 1.49, p = 0.787, vs. shuffle: t = 14.12, p = 10−4). (d) Example 3-d manifold (left) and corresponding 2-d flow field (right). (e) Example of across-day manifold alignment. Manifolds are constructed using all available neurons of each day. (f) Across-day predictions as in (c) but using aligned manifolds (struggling/immobility: d3: vs. reference: t = 0.55, p = 1, vs. random: t = 21.53, p = 10−5, vs. shuffled: t = 12, p = 2 * 10−4, d9: vs. reference: t = 1.18, p = 0.872, vs. random: t = 18.51, p = 2 * 10−5, vs. shuffle: t = 8.43, p = 0.001; correlation of speed: d3: vs. reference: t = 1.36, p = 0.70, vs. random: t = 28.66, p = 3 * 10−6, vs. shuffle: t = 20.31, p = 2 * 10−5, d9: vs. reference: t = 2.54, p = 0.155, vs. random: t = 52.41, p = 10−7, vs. shuffle: t = 35.24, p = 10−6). (b, e) Dashed lines: chance level. One-way repeated measures ANOVAs followed by paired t tests with Bonferroni correction, n = 6 mice. *p https://doi.org/10.5281/zenodo.10378756.</p