Fear and safety engage competing patterns of theta-gamma coupling in the basolateral amygdala

Abstract

Theta oscillations synchronize the basolateral amygdala (BLA) with the hippocampus (HPC) and medial prefrontal cortex (mPFC) during fear expression. The role of gamma-frequency oscillations in the BLA is less well characterized. We examined gamma- and theta-frequency activity in recordings of neural activity from the BLA-HPC-mPFC circuit during fear conditioning, extinction, and exposure to an open field. In the BLA, slow (40-70 Hz) and fast (70-120 Hz) gamma oscillations were coupled to distinct phases of the theta cycle and reflected synchronous high-frequency unit activity. During periods of fear, BLA theta-fast gamma coupling was enhanced, while fast gamma power was suppressed. Periods of relative safety were associated with enhanced BLA fast gamma power, mPFC-to-BLA directionality, and strong coupling of BLA gamma to mPFC theta. These findings suggest that switches between states of fear and safety are mediated by changes in BLA gamma coupling to competitive theta frequency inputs.

Figure 1. Two types of BLA gamma frequency oscillations are coupled to theta during fear recall

(A) Experimental protocol. See text for detailed description. (B) Wavelet transform (color plot) of BLA LFP (gray) recorded during recall session (Day 4). Lower traces, slow (40-70 Hz, green) and fast (70-120 Hz, blue) gamma events, occurring at distinct phases of the theta oscillation (black). Boxes indicate representative high-amplitude gamma events in each frequency band. (C) Phase-Amplitude comodugram of a representative BLA LFP recording demonstrating modulation of high frequency power (y-axis) by low frequency oscillation phase (x-axis). Warm colors indicate stronger modulation; note the prominent modulation of separate slow (40-70 Hz) and fast (70-120 Hz) gamma peaks. (D) Histograms for the occurrence of slow gamma troughs, fast gamma troughs and multi-unit spikes (top three panels) and the preferred phase of significantly phase-locked (p < .05, Rayleigh test) multi-units (n=48) and single units (n=38; bottom two panels) relative to phases of the theta (4-12 Hz) oscillation. Error bars, here and throughout, are +/− SEM, except as otherwise noted.

Figure 2. BLA theta-gamma coupling increases during conditioned fear

(A) Example power spectrum of BLA LFP during pretone (black), an aversive CS+ (red), and a neutral CS− (blue). Presentation of an aversive CS+ elicits higher BLA theta power (peak at 6Hz). (B) Example comodugrams of theta–gamma coupling during pretone (left; 30s before tone), CS+ (right; during tone), and shift predictor of CS+ data (middle). (C) Mean theta-fast gamma coupling strength for CS+ (red) and shift predictor (gray) normalized to pre-tone (black line at 0, n=15). Significance lines (top): CS+/pretone (black) and CS+/shift predictor (gray) differences (p < 0.05/21, Bonferroni-corrected, sign-rank). (D) Mean theta-fast gamma coupling strength as a function of instantaneous theta frequency (n=15). Significance lines (top): Gray (uncorrected, p < 0.05) and black (Bonferroni-corrected sign rank, p < 0.05/15). (E) Change in theta-gamma coupling strength from pretone to CS+ for fast gamma (blue) and slow gamma (green) as a function of instantaneous theta frequency (left) and averaged across the theta range (right). Significance lines (top): Differences from pre-tone for each gamma frequency band. Light blue (uncorrected) and dark blue (Bonferroni-corrected, sign-rank, p<0.5/15). (F) Theta phase-fast gamma amplitude coupling strength as a function of theta power, binned in multiples of SD from the mean of the pretone theta power. * p < .05, ** p < .01, *** p <.001, sign-rank.

Figure 3. BLA fast gamma power decreases during conditioned fear

(A) Example multi-taper spectrograms of BLA LFP during a CS+ (top) and CS− (bottom) presentation. Power was normalized by z-scoring in each frequency range. Black lines, stimulus onset and offset. (B) Fast gamma power during CS+ (red) and CS− (blue) presentations for Discriminators (D) and Generalizers (G). ** p <.01, sign-rank. (C) The difference between CS− and CS+ fast gamma power plotted by animal, as a function of discrimination score (CS+ - CS− percent freezing), with Pearson's r and p-value indicated. Grey box spanning panels (B) and (C) indicates data from the discriminator group. (D) Fast gamma power (top) and theta-fast gamma coupling strength (bottom) as a function of freezing on a trial-by-trial basis for an example animal. Each symbol represents data from a single trial. Data are normalized to pretone values. (E) Population data showing fast gamma power (black) and theta-fast gamma coupling (purple) as a function of freezing level (p<.001 and p<.05, respectively, MLR). All data is mean-normalized.

Figure 4. Increased fast gamma power during the CS− reflects synchronous neural firing

(A) Left, histogram of the preferred fast gamma phases for significantly (dark blue) and non-significantly (light blue) phase-locked multi-unit recordings (p < .05, Rayleigh test). Black line is a cartoon depiction of fast gamma oscillation phases. (B) Percentage of multi-units significantly phase-locked to the fast gamma oscillation, compared to shift predictor. *** p < .001, McNemar's test. (C) Left, pie charts illustrating the percentage of recordings demonstrating significant phase-locking to fast gamma during the CS+ only (red), CS− only (blue), or both (magenta) in Discriminators (top) and Generalizers (bottom). Right, percentage of significantly phase-locked units to CS+ and CS−, including overlap. * p < .05, McNemar's test. (D) Change in multi-unit phase-locking strength to fast gamma from CS+ to CS− for discriminators (black, D) and for generalizers (grey, G). Mean change in discriminators is significantly different from 0 (** p < .01, sign-rank) and greater than in generalizers (** p<.01, rank-sum).

Figure 5. A subset of BLA single units synchronize with BLA fast gamma

(A) Left, fast gamma trough-triggered firing rate of an example single unit. Blue line, trough-triggered LFP. Right, distribution of spikes by gamma phase for this unit. Blue arrow indicates preferred phase. (B) Histogram of the preferred fast gamma phases for significantly (blue) and non-significantly (gray) phase-locked single units (p < .05, Rayleigh test for both distributions). Oscillatory cycle is repeated for clarity. (C) Spike distribution as a function of fast gamma power for significantly phase-locked cells (blue) and all other cells (gray). Fast gamma power was positively correlated with spike rate of both phase-locked (r=0.5510, p=4.5 × 10⁻⁶, MLR) and other cells (r=0.2048, p=0.0011, MLR), but this relationship was significantly stronger for phase-locked units (inset: phase-locked, r=0.35+/−.09; others, r=0.18+/−.04; p=.0232, rank-sum). (D) Trial-by-trial firing rate as a function of freezing rate for an example fast gamma phase-locked unit (r=-.7729, ** p < .01). Gray arrow indicates mean pretone firing rate. (E) Pretone-normalized firing rate as a function of mean-normalized freezing level averaged across all phase-locked (blue) and other (gray) single units. A significant effect of freezing was seen only on phase-locked cells (p < .001, MLR). (F) Change in firing rate from low- to high-freezing trials for phase-locked (blue) and other (gray) single units. Decrease in rate for phase-locked units was significantly different from both 0 (p < .05, one-sample t-test) and from that in other units (p < .05, unpaired t-test).

Figure 6. Synchrony and directionality of fast gamma in mPFC-BLA-vHPC circuit

(A) Top, BLA fast gamma trough-triggered LFPs from mPFC (black), BLA (green) and vHPC (purple). Bottom, fast-gamma trough-triggered spectral coherence for specific region pairs. (B) Left, fast gamma power in the mPFC (top) and vHPC (bottom) during the CS+ (red) and CS− (blue) in discriminators (D) and generalizers (G). ** p < .01, sign-rank. Right, difference in fast gamma power between CS− and CS+ as a function of discrimination score for mPFC (top) and vHPC (bottom). (C) Left, probability (over time) of observing near zero phase synchrony in the fast gamma range by CS type, for discriminators and generalizers. Right, ratio of probability by CS type, as a function of discrimination score. Each symbol represents data from an individual animal. (D) Mean fast gamma Granger Causality Index for the mPFC-BLA (top), BLA-vHPC (middle), and vHPC-mPFC (bottom). Green, BLA lead; gray, mPFC lead; cyan, vHPC lead. *p<0.05, sign-rank. (E) Difference in PFC to BLA Granger lead strength (see text) between CS− and CS+, as a function of discrimination score. (F) Schematic of predominant directionality of fast gamma between mPFC, BLA, and vHPC, inferred from the data presented in D. A safety signal from the mPFC is propagated to the BLA, synchronizing fast gamma activity within the mPFC-BLA circuit.

Figure 7. Increased BLA fast gamma is associated with mPFC-to-BLA theta directionality

(A) Mean CS+-evoked theta phase modulation of gamma frequency activity in the BLA (left, n=23), mPFC (middle, n=27), vHPC (right, n=17). (B) CS+evoked phase-locking (MRL) of BLA fast gamma with its local theta oscillation (green) compared to BLA fast gamma phase locking with mPFC theta (grey, top panel), vHPC theta (cyan, middle panel), and dHPC theta (bottom panel). **p<.01, signrank. (C) The number of BLA multi-unit recordings significantly phase-locked (p<.05/4, bonferroni corrected) to fast gamma oscillations in the mPFC (gray), BLA (green), vHPC (blue), mPFC and BLA (gray/green), BLA and vHPC (green/blue), and all structures (black). All recordings that phase-locked to the dHPC gamma oscillation (2%) also phase-locked to the vHPC gamma oscillation and were thus included with the vHPC in this depiction. (D) Fast gamma power in the BLA, as a function of the percentage of time windows in which the BLA theta leads mPFC theta (top), or mPFC theta leads BLA theta (bottom). Data are from a representative animal; each symbol represents data from single trial. (E) Population averages quantifying BLA fast gamma power for periods when instantaneous theta directionality corresponds to a BLA lead (green), no lead (black), or mPFC lead (gray). (F) Gamma power as a function of the relative strength of coupling of BLA gamma to mPFC vs BLA theta (z-scored relative to BLA theta values).

Figure 8. mPFC lead and BLA fast gamma power in extinction and the open field

(A) Freezing values for mice undergoing extinction during extinction training (CS+ data only). (B) Top, power spectrogram of BLA LFP from a representative animal, showing trial to trial changes in fast gamma power through extinction. Bottom, population mean +/− SEM fast gamma amplitude through extinction for BLA (green) and mPFC (gray). (C) Mean +/− Granger causality index, normalized by pretone value, for mPFC→BLA (gray) and BLA→mPFC (green) directions as a function of trial number. GCI^mPFC→BLA significantly increased throughout extinction (p=4.7 × 10⁻⁵, MLR), without a corresponding change in the GCI^{BLA →mPFC} (p=.97). Inset, relative mPFC granger lead strength (see text) from R1 to E10. (D) Representative paths (yellow) of an anxious (left) and a non-anxious (right) mouse during exploration of a novel open field. Data from center (red), periphery (blue), and transition (gray) epochs was analyzed separately. (E) Fast gamma power by open field zone for anxious (n=9, left) and non-anxious (n=6, right) mice. (F) mPFC Granger lead strength by open field zone for anxious (left) and non-anxious (right) mice.