Analysis of electrohippocampographic EEG recordings

EEG data were exported to NeuroScore 3.2.9306-1 (Data Sciences International, DSI, USA) for further Fast-Fourier Transformation (FFT) based frequency analysis in the range of 0.5–500 Hz, including the following distinctive frequency bands: delta 1 (δ₁, 0.5–4 Hz), delta 2 (δ₂, 1–4 Hz), theta 1 (θ₁, 4–8 Hz), theta 2 (θ₂, 4–12 Hz), alpha (α, 8–12 Hz), sigma (σ, 12–16 Hz), beta 1 (β₁, 12–30 Hz), beta 2 (β₂, 16–24 Hz), beta 3 (β₃, 16–30 Hz), gamma low (γ_low, 30–50 Hz), gamma mid (γ_mid, 50–70 Hz), gamma high (γ_high, 70–100 Hz), gamma ripples (γ_ripples, 80–200 Hz), and gamma fast ripples (γ_{fast ripples}, 200–500 Hz)^91–93. Note that we have included also a broader theta frequency band (theta-alpha band, θ₂) in our analysis, based on the complex functional interdependence of hippocampal oscillatory activity^{12,13,102–104}.

For FFT based analysis, the duration of the individual EEG epochs was determined as 2 s^91–93. Mean relative EEG power (%) of the individual frequency ranges was calculated for the individual circadian stages, i.e., two dark cycles (DC1, DC2, 12 h each) and two light cycles (LC1, LC2, 12 h each), and 6 h post urethane 1 and 2 injection phases (U1, U2). Potential EEG artefacts were identified by both manual inspection of the EEG and the automated artefact detection tool of Neuroscore and were eliminated for EEG relative power analysis^91,92,94.

Relative activity counts and temperature data were also analyzed for baseline (R1, R2) and post urethane recordings (U1, U2) mentioned above. Importantly, activity data (active state, i.e., activity units > 0, or inactive state, i.e., activity units = 0) during the conventional 12 h/12 h light/dark cycle (starting at 5:00 a.m.) were correlated with the relative EEG power of the individual frequency bands from the hippocampal CA1 deflection.

Data were statistically analyzed and displayed as mean ± SEM. Statistics for FFT based frequency analysis were performed using multiple Student’s t-test, corrected for multiple comparison using the Holm-Sidak approach (*p < 0.05; **p < 0.01; ***p < 0.001). Statistics and graphical representations were conducted using GraphPad Prism 6 for Windows (Graphpad Software, Inc., USA).