Abstract
Successfully differentiating safety from danger is an essential skill for survival. While decreased activity in the medial prefrontal cortex (mPFC) is associated with fear generalization in animals and humans, the circuit-level mechanisms used by the mPFC to discern safety are not clear. To answer this question, we recorded activity in the mPFC, basolateral amygdala (BLA) and dorsal and ventral hippocampus in mice during exposure to learned (differential fear conditioning) and innate (open field) anxiety. We found increased synchrony between the mPFC and BLA in the theta frequency range (4–12 Hz) only in animals that differentiated between averseness and safety. Moreover, during recognized safety across learned and innate protocols, BLA firing became entrained to theta input from the mPFC. These data suggest that selective tuning of BLA firing to mPFC input provides a safety-signaling mechanism whereby the mPFC taps into the microcircuitry of the amygdala to diminish fear.
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Acknowledgements
We would like to thank T. Spellman and other members of the Gordon laboratory for technical assistance and discussions. This work was supported by grants from the US NIMH to J.A.G. (R01 MH081968 and P50 MH096891) and E.L. (F32 MH088103), by the International Mental Health Research Organization (J.A.G.) and by the Charles H. Revson Foundation (E.L.). J.M.S. is supported through the Columbia University Medical Scientist Training Program.
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E.L. designed and performed the experiments, analyzed the data and wrote the paper. J.M.S. analyzed the data. M.A.T. assisted in performing the experiments. A.Z.H. assisted in analyzing the data. J.A.G. designed the experiments, supervised the performance of the experiments and data analysis, and wrote the paper.
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Supplementary Figure 1 Freezing levels throughout the test session
(a) Distribution of the changes in percent freezing between the CS- and CS+. Animals showed generalized freezing to the CS- up to 10% more than the CS+. Thus, 10% more freezing to the CS+ than the CS- was judged to also be generalization (grey area). The threshold for discrimination was chosen as freezing to the CS+ at least 10% more than the CS-.(b) Percent freezing on each trial of the test session (Day 4) in Generalizers (b) and Discriminators (c). Note that minimal extinction occurs during the test session (percent freezing on the first versus last CS+ (n=17, 56.9+/- 5.61% vs 47.6 +/-4.7%, signrank, p>0.05) and CS- (first trial, 35+/- 4.98%, last trial, 26+/-5.11%, ranksum, p>0.05), mean +/- s.e.m, p ≥ 0.05, signrank test).
Supplementary Figure 2 Electrode placements and obtained recordings
(a) Examples of electrode tracks in the BLA, mPFC, vHPC and dHPC. Arrows point to lesions at the end of electrode tracks. (b) An example of local field potentials recorded at all sites and a single unit recorded in the BLA during five pip presentations of the CS+ (red vertical lines).
Supplementary Figure 3 vHPC and dHPC pip-evoked theta power does not differ between stimuli for generalizers or discriminators
(a) Group data showing pip-induced theta power. vHPC: Generalizers (n=10), CS+,1.05±1.07, CS-,1.03±1.05, signrank, p>0.05, Discriminators: (n=9),CS+, 1.04±1.05, CS-, 1.02±1.05, signrank, p>0.05; dHPC: Generalizres (n=8), CS+, 0.93±10.11, CS-, 0.89±10.05, signrank, p>0.05. Discriminators(n=7), CS+, 0.85±1.03, CS-, 0.89±1.02, signrank, p>0.05. (b) Change in BLA (multiple comparisons ANOVA, p<.01) and mPFC power (multiple comparisons ANOVA, p=.06) from CS- to CS+ is greater in discriminators than generalizers. No significant differences between groups were seen in vHPC and dHPC.
Supplementary Figure 4 Theta power is modulated by freezing, not velocity
(a,b) Examples of BLA and mPFC power spectra in Generalizers (a) and discriminators (b) during immobility (upper) and mean power increases from pre-tone (lower) by animal for immobility. The same increase in modulation of CS+ theta power relative to CS- theta power is seen during immobility as throughout the session. * p < 0.05, paired signrank. Note that when the analysis is restricted to immobility, theta power modulation is most prominent at the lower frequencies of the theta band (4 – 8 Hz). (c-d) BLA and mPFC theta power (c) and coherence (d) do not change with speed. (e) mPFC and BLA theta power changes are correlated with each other (n=22, r=0.7, p=2.8×10-4, spearman correlation).
Supplementary Figure 5 BLA activity during differential fear conditioning
(a) CS+/CS− differences in BLA-mPFC coherence are larger in Discriminators than in Generalizers (Wilcoxon ranskum, p<.01). (b) BLA firing rates increase with CS presentation (mean +/− s.e.m.; yellow bar, pip duration). (c) Generalizers have bidirectional information flow between the mPFC and BLA during stimulus presentation. Color plots are phase locking strength as a function of lag for all multiunit recordings from Generalizers during the CS+ (left) and CS- (right) aligned by peak lag and grouped by significance of phase-locking (cool colors, n.s.; warm colors, p < 0.05). Histograms below each plot are distributions of lags at which peak phase-locking occurred for significant units only (CS+; n=19, signrank, p>0.05, CS-; n=14, signrank, p>0.05). (d) For Generalizers, an mPFC lead in theta power changes is not correlated with freezing levels on a given trial.
Supplementary Figure 6 Behavior and recordings during exposure to the open field
(a) Experimental procedure. Animals were exposed to a neutral familiar environment for 4 days. On the 4th day, the animals were also exposed to a novel, brightly lit open field.(b) Distribution of percent center time in our cohort. Vertical line (at 10% center time) shows where we separated the Anxious animals, concentrated to the left of the line, and the Non-anxious animals, to the right of the line. (c) LFP recordings (grey) from the BLA, mPFC, vHPC and dHPC were filtered for theta (red lines) for the analysis. Multiunit firing was recorded in the BLA
Supplementary Figure 7 BLA-mPFC synchrony increases in the open field relative to the first exposure of the familiar environment
Significant change in theta power correlations from the first exposure to the Familiar Environment to the Open Field (left, R= − 0.592, p< 0.05); quantification of average theta power correlation changes from Familiar Day 1 to the Open Field and Familiar Day 4 to the Open Field (right).
Supplementary Figure 8 Non-anxious mice show no changes in BLA-mPFC synchrony during transitions and no net directionality in BLA firing relative to the mPFC
(a) BLA-mPFC coheregrams around the transition points into (top) and out of (bottom) the center of the open field. Coherence +/-2sec around the transitions is normalized to 3 seconds of baseline (-5 to -2 pre-transition). (b) Average BLA (blue, solid line) and mPFC (black, solid line) theta power +/- sem (faded bands) around the transitions into (top) and out of (bottom) the center, normalized to 3 seconds of baseline (-5 to -2 pre-transition). (c) Colorplots show strength of BLA multiunit phase locking to mPFC theta in the periphery (left) and the center (right) of the open field as a function of lag for the Non-anxious mice (n=13 stereotrodes from 6 mice, cool colors, n.s.; warm colors, p < 0.05). Histograms show the distributions of peak phase locking lags. Only the significantly phase locked units are shown.
Supplementary Figure 9 Theta phase reset is sharper during a stimulus recognized as aversive
(a) Individual (black lines) and averaged (beige line) theta-filtered BLA LFP responses to CS- (left) and CS+ (right) pips. (b) Distributions of theta phases at 20 ms after each pip in BLA and mPFC LFPs from an example Discriminator. (c) Mean +/- s.e.m. of theta-phase distribution peak half-width by stimulus type (red, CS+, blue, CS–). Higher half-width indicates wider distribution (weaker phase-reset). BLA: Generalizers, CS+ 231.84±115.33, CS–, 225.92±116.88, paired signrank, p>0.05; Discriminators, CS+,228.48±111.32, CS–, 259.24±19.72, paired signrank, p<.01; mPFC: Generalizers, CS+, 209.57±116.37, CS–,214.24±112.85, paired signrank, p>0.05; Discriminators, CS+, 211.40±110.13, CS– 251.66±116.43, paired signrank test, p<.01.
Supplementary Figure 10 Schematic for proposed BLA-mPFC interactions in learned fear and innate anxiety during safety (left) and danger (right)
mPFC entrains BLA activity to its theta oscillations when Discriminators hear a CS- during learned fear and when Anxious animals are in the periphery during a test of innate anxiety (left panel). mPFC-BLA theta communication is bi-directional during recognized Danger (right panel) during learned fear (CS+ for all animals and CS- for Generalizers) and during innate anxiety (center of the open field for Anxious animals).
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Likhtik, E., Stujenske, J., A Topiwala, M. et al. Prefrontal entrainment of amygdala activity signals safety in learned fear and innate anxiety. Nat Neurosci 17, 106–113 (2014). https://doi.org/10.1038/nn.3582
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DOI: https://doi.org/10.1038/nn.3582
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