Vigilance-Associated Gamma Oscillations Coordinate the Ensemble Activity of Basolateral Amygdala Neurons

Abstract

Principal basolateral amygdala (BL) neurons profoundly influence motivated behaviors, yet few of them are activated by emotionally valenced stimuli. Here, we show that a likely explanation for this paradox is the synchronizing influence of the high-gamma rhythm. High-gamma (75-95 Hz) entrained BL firing more strongly than all other rhythms. It was most pronounced during states of increased vigilance, when rats were apprehensive. Relative to behavioral states, high-gamma produced minor changes in firing rates yet dramatic increases in synchrony. Moreover, connected pairs of cells showed similarly high levels of entrainment and synchronization. Unexpectedly, prefrontal- and accumbens-projecting cells, respectively, showed high and low entrainment by high-gamma, indicating that this rhythm differentially synchronizes the activity of BL neurons projecting to specific sites. Overall, our findings suggest that individual BL neurons encode information not only by changing their firing rates, but also by synchronizing their collective activity, amplifying their impact on target structures.

Keywords: amygdala; beta; connectivity; emotions; fear; memory; oscillations; synchrony; theta.

Fig. 1

Behavioral tasks and changes in theta, beta, and gamma power seen during the foraging task. (A,B) Behavioral tasks. (A1–2) Foraging task. (A1) Apparatus. (A2) Five example trials where red dots indicate rat position and the distance between the dots is proportional to the rat’s velocity. S, A, and F mark successful, aborted and failed trials, respectively. Empty black squares on the right indicate position of food pellets. Vertical dashed lines mark 25 cm intervals. (B) Shuttle task. (B1) Apparatus. (B2) Three example trials where red and blue dots indicate rat position and the distance between the dots is proportional to the rat’s velocity. (C,D) Spectrograms showing z-scored power fluctuations in various frequency bands as a function of relative time. All spectrograms shown here and in the following figures were normalized using the same data (see Methods) and thus can be directly compared. Warmer colors indicate higher power. (C–D) Spectrograms (top) and average unit activity (bottom; normalized to baseline firing rates in C1) in various conditions (black, principal cells; red, interneurons; dashed and solid lines respectively represent cells with significantly decreased [Type-1] and increased [Type-2] firing rates during foraging (Kruskal-Wallis ANOVA followed by Dunn test, p<0.05). (C1) All trials with foraging (1238 trials). (C2) Subset of trials where rats, after hesitating at the door retreated in the nest (134 trials). Post-hoc Dunn tests on the data shown in C1 revealed that theta power was significantly (p<0.05) different in the four phases (baseline<waiting<foraging<escape). The same approach revealed that beta power was significantly different in the four phases (foraging<waiting<escape<baseline). For mid-gamma, power was higher during baseline relative to the other three phases with no difference between waiting, foraging, and escape. For low- and high-gamma, power differed significantly in the four phases (foraging>waiting=escape>baseline). (D) Subsets of trials that followed failed (D1) or successful (D2) trials. Kruskal-Wallis ANOVAs; df=5; low-gamma X²=430.8; p<0.001; high-gamma X²=552.2; p<0.001. Post-hoc Dunn tests revealed the following significant (p<0.05) differences. For low-gamma, (foraging [n-1,fail]=foraging[n-1,success]) > (baseline[n-1,fail]=waiting[n-1,fail]) > waiting[n-1,success] > baseline[n-1,success]. For high-gamma, (foraging [n-1,fail]=waiting[n-1,fail]= baseline[n-1,fail]) > foraging[n-1,success] > waiting[n-1,success] > baseline[n-1,success]. Number of units in the various panels: (C1, D1, D2: principal cells, 271 Type-1 and 25 Type-2; interneurons, 11 Type-1 and 35 Type-2; C2 principal cells, 226 Type-1 and 22 Type-2; interneurons, 8 Type-1 and 29 Type-2). Average baseline firing rates: Principal cells Type-1 (0.21 ± 0.03 Hz) and Type-2 (1.55±0.33 Hz); Interneurons Type-1 (27.47±3.45 Hz) and Type-2 (22.44±2.19 Hz). See also Figure S1 as well as Tables S1 and S2.

Fig. 2

Relation between running speed and gamma power. (A,B) Spectrograms showing z-scored power fluctuations in various frequency bands as a function of when the rats’ velocity during foraging was low (A1; 30% trials with lowest velocity; n=371), when it was high (A2; top 30% trials; n=371), or when rats hesitated during foraging, exhibiting one or more starts (B1) and pauses (B2). (C1) Relation between rat velocity and LFP power of different frequencies as estimated by a generalized linear model. Beta velocity values are color coded (scale on left) during foraging. (C2) Average beta velocity values (y-axis) computed from entire foraging period for different power in different frequencies. (C3) Bins of C1 with statistically significant positive (red) and negative (blue) beta values during foraging (relative time, x-axis). (C4) Proportion of bins with significant beta velocity values during entire foraging period. Repeated measures Friedman ANOVAs relating absolute beta values associated to the speed, predator, and prior trial variables for low and high gamma: X²s=417 and 582, df=2, p<0.001. Post-hoc Dunn tests for GLM results revealed the following significant differences (p<0.05): absolute beta speed > absolute beta prior trial > absolute beta robot for both low- and high-gamma. (D) Spectrogram showing z-scored power fluctuations in various frequency bands as a function of relative time in the shuttle task (277 trials).

Fig. 3

Entrainment of BL firing by different LFP rhythms. (A–B) Peri-event histograms of neuronal discharges for a representative principal cell (A) and fast-spiking interneuron (B) around large amplitude (≥2 SD) oscillatory peaks in different frequency bands (1, high-gamma; 2, low-gamma; 3, beta; 4, theta). In all cases, the bin width was set to 0.1 of the period under consideration. (C–D) Entrainment of unit activity (y-axis; median of the entire sample) as a function of frequency (x-axis) for principal cells (C) and fast-spiking interneurons (D). (E) Proportion of principal cells significantly entrained by different rhythms (y-axis) as a function of distance between the recorded cell and reference LFP (x-axis). (F–G) Frequency distributions of entrainment by high-gamma among principal cells (F) and interneurons (G). (H–I) Frequency distribution of preferred firing phase in relation to high-gamma for principal cells (H) and interneurons (I). (J) Average firing phase in relation to high-gamma for principal cells (blue) and interneurons (red).

Fig. 4

Impact of behavioral states on entrainment of unit activity by high-gamma. Average ± SEM firing phase (A) and entrainment (B) by high-gamma for principal cells (A1, B1) and interneurons (A2, B2) during different behavioral states (x-axis; QW, quiet waking; B, baseline; W, waiting; F, foraging). Principal cells (C) and interneurons (D) were sorted by their entrainment in quiet waking (C1, D1) and high-gamma bursts were stratified by amplitude (y-axes). Data obtained in the other states (C2–4, D2–4) was plotted without changing the ordering of the cells. Warmer colors indicate stronger entrainment. Note that the number of cells included in panels A (54 principal cells and 45 interneurons) vs. B–D differs because panel A does not include cells that were not significantly entrained whereas panels B–D do (65 principal cells and 50 interneurons). Non-significantly entrained cells were excluded from A since this panel reports on phase, which is meaningless when cells are not entrained. Regarding firing entrainment, a repeated measures Friedman ANOVA revealed minor but significant differences in entrainment magnitude as a function of state in interneurons (X²_(3,199)=10.49; p=0.015) but not in principal cells (X²_(3,259)=4.99; p=0.17). The maximal significant difference in entrainment between states was 4% in interneurons (Post-hoc Dunn tests; p<0.05).

Fig. 5

State-related variations in firing rates are much larger than those produced by high-gamma. Comparisons of GLM beta coefficients for gamma amplitude and foraging (A), waiting (B), and baseline (C). Red, 50 interneurons; Blue, 381 principal cells. The data was analyzed in 12 ms windows but longer windows (50 or 100 ms) yielded qualitatively identical results. On the top right of each panel, we provide the average absolute ratio of the beta coefficients associated with state and gamma.

Fig. 6

Impact of high-gamma on firing rates. (A) Principal cells. (B) Interneurons. Examples of PEHs of neuronal discharges computed around high gamma peaks (≥ 2 SD) for principal cells (A1–4, A6) and interneurons (B1,3–6). Firing entrainment (y-axis) as a function of change in firing rate (% of baseline) produced by high-gamma for principal cells (A5) and interneurons (B2). Firing rate (FR) changes are expressed as (FR(γ) – FR(noγ)) / FR(noγ). Related to Figure S2.

Fig. 7

Impact of high-gamma on firing synchrony. Couples of principal cells (1), interneurons (2), or principal cells and interneurons (3). (A) Crosscorrelation of unit activity when high-gamma power was high (blue) or low (red). Average ± SEM of normalized CCGs for all available cell couples. Note that in contrast with couples of principal cells (A1) and interneurons (A2), mixed couples generally fired with a time lag between them, interneuronal discharges following principal cells’ by ~2 ms (A3). Thick lines, averages; thin lines, SEM. (B) Synchronization indices for individual cell couples where both neurons generated at least one spike in at least one of the analyzed time windows. The n’s indicate the number of couples included in each analysis but we only show in B1 and B3, we only show the 200 most active cell pairs for clarity. Because principal cells fire rarely, only ~40% of cell couples met this criterion. In contrast, it was met by all interneuron pairs as well as most mixed couples. In B1–3, the black horizontal lines in the blue rectangles indicate the medians. The black vertical lines indicate the range of values including 95% of the observations. The top and bottom edges of the blue rectangles indicate the 75^th and 25^th percentile, respectively. In A and B, the distance between recorded units was 200 μm. (C) Relation between synchronization index (y-axis; average ± SEM) and distance between units (x-axis) when high-gamma power was high (blue) or low (red). In B and C, significance levels are indicated, based on signed-rank tests. Provided we controlled for gamma level, we found no effect of states (QW, baseline, waiting, foraging) on SIs in pairs of interneurons (ANOVA F_(3,1223)=0.87, p=0.45), and couples of principal cells and interneurons (ANOVA F_(3,6057)=1.0, p=0.39). This analysis could not be performed in pairs of principal cells, due to their low firing rates. (D) Relation between R product and gamma-related firing synchrony. Cell couples were stratified by their R product (numbers on the right are percentiles; thick lines are averages; thin lines are SEM). Related to Figures S2.

Fig. 8

Relation between connectivity and entrainment by high-gamma. (A) Connections from principal cells to interneurons separated by 200 μm. (A1) Red and blue are used to represent mono-synaptically connected vs. non-connected cell couples, respectively. The x- and y-axes represent the resultant vector of the two cells in each couple. The graphs on the left and at the bottom plot the probability of connections as a function of the resultant vector. To generate these graphs, we computed the probability of connections in bins containing 20% of the cells. The black lines indicate the borders of the bins. (A2) Average unit-unit coherence (±SEM, dotted lines) plotted as a function of frequency for connected (red) and unconnected (blue) couples of principal cells and interneurons. (B) Entrainment of principal cells by high-gamma varies depending on the cells’ projection site(s). B shows frequency distributions of the resultant vector for all presumed principal cells (n = 381; B1), all cells that were positively identified as projection cells by antidromic invasion (n = 56; B2), cells that were backfired from nAc (n = 23; B3), and cells that were backfired from the mPFC (n = 27, B4). Six neurons that were backfired from nAc and mPFC are not included in B3,4. Related to Figure S4, S5, S6, S7, and S8.