Acute Stress Affects the Expression of Hippocampal Mu Oscillations in an Age-Dependent Manner

Abstract

Anxiolytic drugs are widely used in the elderly, a population particularly sensitive to stress. Stress, aging and anxiolytics all affect low-frequency oscillations in the hippocampus and prefrontal cortex (PFC) independently, but the interactions between these factors remain unclear. Here, we compared the effects of stress (elevated platform, EP) and anxiolytics (diazepam, DZP) on extracellular field potentials (EFP) in the PFC, parietal cortex and hippocampus (dorsal and ventral parts) of adult (8 months) and aged (18 months) Wistar rats. A potential source of confusion in the experimental studies in rodents comes from locomotion-related theta (6-12 Hz) oscillations, which may overshadow the direct effects of anxiety on low-frequency and especially on the high-amplitude oscillations in the Mu range (7-12 Hz), related to arousal. Animals were restrained to avoid any confound and isolate the direct effects of stress from theta oscillations related to stress-induced locomotion. We identified transient, high-amplitude oscillations in the 7-12 Hz range ("Mu-bursts") in the PFC, parietal cortex and only in the dorsal part of hippocampus. At rest, aged rats displayed more Mu-bursts than adults. Stress acted differently on Mu-bursts depending on age: it increases vs. decreases burst, in adult and aged animals, respectively. In contrast DZP (1 mg/kg) acted the same way in stressed adult and age animal: it decreased the occurrence of Mu-bursts, as well as their co-occurrence. This is consistent with DZP acting as a positive allosteric modulator of GABA_A receptors, which globally potentiates inhibition and has anxiolytic effects. Overall, the effect of benzodiazepines on stressed animals was to restore Mu burst activity in adults but to strongly diminish them in aged rats. This work suggests Mu-bursts as a neural marker to study the impact of stress and DZP on age.

Keywords: Mu-rhythm; aging; hippocampus; stress; synchronized oscillation.

Figure 1

(A) Stress protocol: 60 min at rest, followed 1 day later by 60 min under stress on an elevated platform (EP). A cold light source (100 lux) was applied at a distance of 10 cm in front of the rat’s nose to keep the eyes of the animal wide open and its head held up. (B) Representative traces of the Z-scored extracellular fields potential (EFP) simultaneously recorded from the same animal in the prefrontal cortex (PFC), dorsal hippocampus (dHPC) and ventral hippocampus (vHPC) at rest. Raw traces are plotted in gray and filtered (7–12 Hz range) traces are overlaid in black. (C) Spectral analysis of the EFP recorded at rest for each structure (black) and under acute stress (red: PFC n = 13, blue: dHPC n = 13, purple: vHPC n = 12). The top right insert represents the averaged relative change, expressed in percentage of variation. Horizontal dashed line at zero indicates no change. Data are presented as mean ± standard error of the mean (SEM) and shaded area indicates SEM. (D) Coherence for PFC-dHPC, PFC-vHPC and dHPC-vHPC at rest (black) and under stress (orange). In top right insert, averaged relative change expressed in percentage of variation. Horizontal dashed line at zero indicates no change. Data are presented as mean ± SEM and shaded area indicates SEM.

Figure 2

(A) Spectrogram of dHPC at rest (left panel) and under stress (right panel). Note the emergence of intermittent oscillations in the 7–12 Hz range in stress condition. (B) Raw trace of the dHPC and its behavioral correlate. Note the prominent increase in the raw signal. Most of the Mu-bursts events were associated with whisker twitching (WT) both at the onset and ending. (C) EFP Z-scored trace and time-resolved power spectral density (PSD; using a complex Morlet wavelet transform) during a Mu-burst. Mu-bursts correspond to an oscillation with a dominant frequency peak around 7–12 Hz, together with one to several biological harmonics. (D) Distribution of 7–12 Hz power across time at rest (black) and under stress (blue). The bell curve of the distribution after a log transform reveals a unimodal distribution. Note this another peak appearing under stress, with the rest of the distribution unchanged, corresponding to the Mu-bursts associated with WT. (E) Area under the curve (AUC) computed from the Morlet wavelet transform (averaged over the 7–12 Hz range) that reflects the overall amplitude of Mu-bursts. The Mu-bursts AUC increased significantly in stress condition (***p < 0.001, n = 13).

Figure 3

(A) Typical examples of Mu-bursts in Z-scored EFP traces (top) and corresponding time-resolved PSD (bottom) in both the PFC (left) and dHPC (middle), as well as the superposition of these traces (top right) and the time-resolved coherence between the PFC and dHPC (bottom right). These Mu-bursts consisted in oscillations at the same frequencies (7–12 Hz). (B) Extraction of discrete Mu-bursts: (i) raw EFP (red: PFC; blue: dHPC) was wavelet-transformed in the 7–12 Hz range, averaged over frequency and smoothed across time (Kalman filter), resulting in the black trace. Mu-bursts starts and stops were determined using a double threshold, one for the onset (blue line) and another for the completion (red line), also constrained by a burst duration greater than 3 s. (C) Top: Venn diagram illustrating the average occurrence of Mu-bursts in each structure and their co-occurrence; Bottom: distribution of time lags between Mu-bursts onsets, from dHPC relative to PFC. Red line corresponds to zero-lag and purple line represents the median lag. (D) Top: superimposed EFP (red: PFC; blue: dHPC) showing both epochs of Mu-bursts and of baseline oscillations. Middle: time-resolved coherence between the PFC and dHPC. Coherence is maximal during Mu-bursts. Bottom: difference in instantaneous phases from the wavelet transforms (phase-shift) of dHPC and PFC, indicating phase-locking during Mu-bursts. (E) Top: square of the absolute value of the wavelet transform when no Mu-bursts occur (“No”), Mu-bursts occur in both the PFC and dHPC (“both”) and only in one of the two structures (“Only PFC”, “Only dHPC”) in two condition (R: rest; S: stress), (***P_rest/***P_stress) Tukey’s honest significant difference (HSD) test indicated statistical difference between the following pairs: (Rest: R₁-{R₂, R₄}; R₂-{R₁}; R₃-{naught}; R₄-{R₁}; Stress: S₁-{S₂, S₄}; S₂-{S₁, S₃}; S₃-{S₂}; S₄-{S₁}). Bottom: phase-locking value), (***P_rest/***P_stress). See Table 2which indicated statistical difference between pairs.

Figure 4

(A) Top: representative traces of the Z-scored EFP in the parietal associative cortex (PAR). Raw traces are plotted in gray and filtered (7–12 Hz range) traces are overlaid in black; Bottom: spectrogram of PAR under stress. (B) Spectral analysis of the EFP recorded at rest structure (black) and under acute stress (brown) N = 5. (C) Venn diagram illustrating the average occurrence of Mu-bursts in dHPC and PAR and their co-occurrence. Note at rest, Mu-burst are observed independently in the two neighboring structures. Under stress condition the total number increased in both structure. Note that all Mu-bursts detected in PAR co-occurred in the dHPC. (D) Distribution of time lags between Mu-bursts onsets, from dHPC relative to PAR. Brown line corresponds to zero-lag and purple line represents the median lag. Note that all Mu-bursts detected in PAR co-occurred in the dHPC. Note that the Mu-burst are observed independently in the two neighboring structures. Rest: 0.53 s median delay, paired sample T-test ***P < 0.001 stress: 0.48 s median delay, Wilcoxon signed rank test ***P < 0.001.

Figure 5

(A) Stress protocol (same as Figure 1) in aged rats. (B) Representative traces of the Z-scored EFP simultaneously recorded from the same animal in the PFC, dHPC and vHPC at rest. Raw traces are plotted in gray and filtered (Mu range) traces are overlaid in black. (C) Spectral analysis of the EFP recorded at rest for each structure and each group. Adults (black) Aged (color); red: PFC n = 10, blue: dHPC n = 9, purple: vHPC n = 5). Differences were observed between the average spectral properties of adults and aged rats at rest, whatever the structure (PFC: χ² = 57.19; ***P < 0.001 Kruskal-Wallis test; dHPC: F_(2,63) = 132.45; ***P < 0.001 One-way ANOVA test; vHPC Kruskal-Wallis test χ² = 41.61 ***P ≤ 0.001) and without specific frequency range. Inserts corresponded at the average relative changes under stress, expressed in percentage of variation. Stress in aged animal decreased the PSD amplitude in all brain structures (Friedman’s test: χ² = 5.15, *P < 0.05 for the PFC; χ² = 15.61, ***P ≤ 0.001 for the dHPC; χ² = 5.53, *P < 0.05 for the vHPC) whatever the band after correction (see text) (D) Coherence for PFC-dHPC, PFC-vHPC and dHPC-vHPC at rest for each group (black: Adults; gold: Aged; PFC-dHPC n = 10, PFC-vHPC n = 5, dHPC-vHPC n = 5). Hippocampal-prefrontal synchrony at rest was significantly different between aged and adults rats, in the PFC-dHPC and PFC-vHPC (Kruskal-Wallis test: χ² = 14.81 ***PCOH_PFC-dHPC < 0.001; χ² = 24.26, ***PCOH_PFC-vHPC < 0.001). Post hoc (Holm’s Bonferroni) test showed that this difference did not implicate any specific band. Top-right insert: relative change after stress, expressed in percentage of variation. Horizontal dashed line at zero indicates no change. Shaded area indicated SEM. Stress decreased dramatically the coherence between PFC and dHPC in aged rats, for all frequency ranges taken separately (χ² = 5.76, *P < 0.05 Friedman’s test followed by post hoc tests (Holm’s Bonferroni), *P_delta = 0.0166 paired-sample t-test; *P_mu = 0.0098 Wilcoxon’s signed rank test; *Pbeta = 0.0137 Wilcoxon’s signed rank test). Significant difference was also found in PFC-vHPC coherence without incrimination of a specific frequency band (χ² = 4.98, *P < 0.05 Friedman’s test), but not in dHPC-vHPC (F_(1,24) = 0.02, P = 0.8766 Two-way ANOVA; ns p > 0.05; *p < 0.05; **p < 0.01, ***p < 0.001).

Figure 6

(A) Spectrogram of dHPC at rest (top) and under stress (bottom). Note the decrease of Mu-bursts under stress condition. (B) Top: Venn diagram illustrating the average occurrence of Mu-bursts in each structure and their co-occurrence and distribution of time lags between Mu-bursts onsets, from dHPC relative to PFC. Red line corresponds to the zero-lag and the purple line represents the median lag. Under stress condition, the total number of Mu-bursts decreased in the dHPC (*p < 0.05, Wilcoxon signed rank test) and in the PFC (*p < 0.05, paired sample T-test). At rest, there was on average no significant delay (median delay = −0.004 s) between PFC and dHPC bursts, while under stress, bursts were detected first in the dHPC (median delay = −0.0781 s, Wilcoxon signed rank test, ***p < 0.001). (C,D) AUC computed from the wavelet transform (left) and the wavelet coherence (right) from the 7–12 Hz range. In aged group, both decreased significantly under stress (black: rest; color: stress). Significant differences were found for AUC (Wilcoxson signed rank test n = 13 ***P_adults < 0.001 and paired sample T-test n = 9**P_aged < 0.001) and for coherence (paired sample T-test **P_adults < 0.01 and **P_aged < 0.01). (E) Top: square of the absolute value of the wavelet transform when no Mu-bursts occur (“No”), when Mu-bursts occur in both the PFC and dHPC (“both”) or only in one of the two structures (“only PFC” and “only dHPC) in two condition (R: rest; S: stress). dHPC PSD remained low in the absence of Mu-burst and during of occurring and co-occurring Mu-bursts only in the rest condition (Kruskal-Wallis test χ² = 15.12, **P_rest < 0.05 and χ² = 5.41, P_stress = 0.1444) Bottom: phase-locking value still remained significantly higher during co-occurring of the Mu-bursts at rest, and during PFC-occurring only (One-way ANOVA F_(3,23) = 7.66, **P_rest < 0.01 and F_(3,22) = 9.57 ***P_stress < 0.001). See table which indicated statistical difference between pairs.

Figure 7

(A) Stress protocol (same as Figure 1) with an acute i.p injection of Diazepam (DZP; 1 mg/kg) vs. vehicle, in adult rats (n = 5) and aged rats (n = 5). (B) Coherence for PFC-dHPC in stress condition, under vehicle (orange) and under DZP (orange red). Top right insert: averaged relative change in coherence, expressed in percentage of variation. Horizontal dashed line at zero indicates no change. Shaded area indicates SEM. A significant decrease was found in the Mu band both for adult (left panel) and aged rats (right panel; *P_adults < 0.01; ***P_aged < 0.001, paired sample T-test). (C) Spectrogram of dHPC under stress for each group after a vehicle or DZP injection. Note the decrease of Mu-bursts under stress condition after an i.p injection of DZP. (D) PFC-dHPC coherence computed from the wavelet transform in the Mu range in three conditions (RV, rest vehicle; SV, stress vehicle; SD, Stress DZP (1 mg/kg)). Adults: COH_PFC-dHPC: Kruskal-Wallis test χ² = 1.63, P = 0.4431 for the PFC-dHPC coherence. Aged: Kruskal-Wallis test: χ² = 6.02, *P < 0.05, followed by paired-sample t-test and Holm-Bonferroni correction *P_RV-SD = 0.0071, *P_RV-SV = 0.0102, *P_SV-SD = 0.0146 for the PFC-dHPC coherence.

Figure 8

(A,B) Power spectrum density of PFC (left) and dHPC (right) EFP recorded at rest with an acute i.p injection of vehicle (black) vs. DZP 1 mg/kg (green), and the averaged relative change due to DZP (insert). Horizontal dashed line at zero indicates no change. Data are presented as mean ± SEM and shaded area indicates SEM. No significant change was observed in this condition, in both age groups. PFC: P_adults = 0.8491; P_aged = 0.2694, paired sample T-test; dHPC: P_adults = 0.4360; P_aged = 0.1473, paired sample T-test. (C) Coherence for PFC-dHPC (adults (left) and aged (right) rats) in rest condition, under vehicle (black) and DZP (green). Top right insert: averaged relative change in coherence, expressed in percentage of variation. Horizontal dashed line at zero indicates no change. Shaded area indicates SEM. No significant differences were found in Mu band in both group (P_adults = 0.2410; P_aged = 0.1159, paired sample T-test).

Figure 9

(A) Coherogram (PFC-dHPC coherence over time) in four conditions (RV; SV; rest-DZP; stress-DZP) for adult rats. Stress enhanced synchronization between PFC-dHPC in 7–12 Hz in the adult group compared to rest. Acute DZP injection reduced this synchrony at rest and under stress. (B) In aged rats, a global decrease (all frequency ranges) was observed under acute stress, but was less pronounced in the 7–12 Hz range. Conversely, DZP alleviated the coherence mainly in the 7–12 Hz. The combination of DZP and stress nearly abolished PFC-dHPC coherence for all frequency ranges.