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. 2018 Oct 11;3(4):437-459.
doi: 10.1002/epi4.12262. eCollection 2018 Dec.

Methodologic recommendations and possible interpretations of video-EEG recordings in immature rodents used as experimental controls: A TASK1-WG2 report of the ILAE/AES Joint Translational Task Force

Affiliations

Methodologic recommendations and possible interpretations of video-EEG recordings in immature rodents used as experimental controls: A TASK1-WG2 report of the ILAE/AES Joint Translational Task Force

Ozlem Akman et al. Epilepsia Open. .

Abstract

The use of immature rodents to study physiologic aspects of cortical development requires high-quality recordings electroencephalography (EEG) with simultaneous video recording (vEEG) of behavior. Normative developmental vEEG data in control animals are fundamental for the study of abnormal background activity in animal models of seizures or other neurologic disorders. Electrical recordings from immature, freely behaving rodents can be particularly difficult because of the small size of immature rodents, their thin and soft skull, interference with the recording apparatus by the dam, and other technical challenges. In this report of the TASK1 Working Group 2 (WG2) of the International League Against Epilepsy/American Epilepsy Society (ILAE/AES) Joint Translational Task Force, we provide suggestions that aim to optimize future vEEG recordings from immature rodents, as well as their interpretation. We focus on recordings from immature rodents younger than 30 days old used as experimental controls, because the quality and correct interpretation of such recordings is important when interpreting the vEEG results of animals serving as models of neurologic disorders. We discuss the technical aspects of such recordings and compare tethered versus wireless approaches. We also summarize the appearance of common artifacts and various patterns of electrical activity seen in young rodents used as controls as a function of behavioral state, age, and (where known) sex and strain. The information herein will hopefully help improve the methodology of vEEG recordings from immature rodents and may lead to results and interpretations that are more consistent across studies from different laboratories.

Keywords: Anesthesia; Awake; Cortical; Minimum standards; Mouse; Ontogeny; Postnatal; Rat; Sleep; Spectral analysis; Spindles; Stereotaxic; Subcortical; vEEG.

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Figures

Figure 1
Figure 1
Wired and wireless telemetry EEG recordings in immature rodents: prolonged continuous recordings with wireless methods and types of artifacts with wired and wireless approaches. AC, Continuous monitoring for 48 h from a P7–P8 rat pup with dam using a wireless approach. Although video‐EEG recordings with either wired or wireless recordings are optimal when performed when the pup is studied in isolation from the dam, wireless recordings can be obtained when the dam is present in the same cage; however, artifacts from the dam are still possible (see text and below). A rat pup was implanted with a wireless telemetry unit at P6 and housed with the dam and littermates in a cage positioned on a receiver base designed for an adult animal. A continuous recording of 48 h was made from a rat pup. Recording from Day 1 is shown in A, and from Day 2 in B; C shows background spontaneous electrical activity in a temporal expansion of part of the record in B (arrow below record in B). Downward arrows in A and B (above the traces) indicate “dropouts” of the EEG signal, which can occur when recordings are performed with wireless telemetry. In the top trace (A), the first 2 arrows mark “dropouts” that were too brief to see at this time scale (see below); however, the last 2 arrows show longer “dropouts” that can be seen as a flat part of the trace.This proof‐of‐concept experiment shows the possibility of conducting nearly continuous recordings—at least for 1–2 days at a time—in immature rats with the dam, but with interruptions of the recording. The rat pup was implanted at P6 (2–4% isoflurane) with a one‐channel miniature wireless telemetry device (Epoch, Epitel, Inc., Salt Lake City, UT). The bandpass of the EEG signals with the wireless recordings was 0.1–120 Hz, 8 dB per octave. DF, Examples of artifacts observed in wired and wireless telemetry recordings of EEG from awake, freely behaving immature rodents. Wired recordings, particularly from immature rodents, are susceptible to movement artifacts that arise from shifting of the connecting wires (D). Wireless telemetric recordings (E and F) can provide a signal with much smaller and fewer artifacts, but wireless recordings are also susceptible to movement artifacts, plus “dropout” artifacts that are shown in an expanded form in F. “Dropouts” in wireless recordings occur occasionally when the transmitter does not properly couple with the receiver (e.g., when the dam blocks transmission between the pup and the receiver). Reprinted with permission from Zayachkivsky et al.30
Figure 2
Figure 2
Examples of EEG/EMG from a male Sprague‐Dawley rat recorded on P7, P10, and P12. Panel A, Wake sleep scoring was done using behavioral (video monitoring), nuchal EMG recordings, and 2 EEG channels recording from the frontal and parietal regions (Right [R.] Frontal‐Left [L.] Parietal; R. Parietal – L. Parietal) through stainless steel screw electrodes. EMG was recorded using 2 subcutaneous stainless steel wires placed over the nuchal muscles (2EEG/1EMG system for EEG/sleep recordings; Pinnacle Technology Inc, Lawrence, Kansas). Wakefulness was characterized by coordinated movements (indicated by green line under EMG), increase in the range of EEG frequencies and their amplitude with age. Quiet sleep (QS) was characterized by lack of EMG activity, low voltage EEG on P7 and appearance of delta (mostly frontal; 1 Hz to <4 Hz rhythms) at P10 and P12 (shown by red line). Increased fast activity is also seen in older ages; included fast alpha/beta rhythms embedded in the delta waves. Active sleep (AS) was indicated by low nuchal EMG activity and presence of EMG bursts during the muscular twitches and jerks (shown by blue *). The range of frequencies was similar in wakefulness and AS. Please note the difference in the type and voltage of activities recorded from the anterior and posterior EEG channels, which helps describe the anteroposterior organization of EEG activities. The insert presents an enlarged version of the delta activity seen in panel A (P10, quiet sleep, R. frontal – L. parietal channel). Panel B, Diagram of the location of the electrodes (stainless steel screw electrodes, red dots; stainless steel EMG wire electrodes, blue lines) on the pup's skull. Acquisition filters were 0.5 Hz (low filter) and 1,000 Hz (high filter). The bar scale indicates the sensitivity and timescale. The figure was provided by Aristea Galanopoulou.
Figure 3
Figure 3
Examples of EEG from a male Sprague‐Dawley rat recorded on P11 and P18. Intermittent vEEG recordings (A) using stainless steel screw electrodes placed at bilateral frontal and occipital regions and referenced with a screw electrode placed over the cerebellum (see diagram of electrode layout in panel B) were done. (A) Examples of awake and asleep (quiet/slow wave sleep and active sleep) EEG studies on P11 and P18, using a referential montage, with a cerebellar reference. The awake background shows a mixture of activities but also the emergence of a 6–7 Hz activity (indicated by the green line), better seen at the frontal leads, which becomes more prominent on P18. Quiet sleep or slow wave sleep shows prominent high amplitude delta activity maximal frontally (red line) with superimposed frontal maximal spindles (blue *). Active sleep shows a fast background and occasional twitches/jerks detected as EMG artifact. Please note the difference in the range of activities recorded at the frontal versus the occipital regions, suggesting that the location of electrodes may alter the recorded signal. Scoring of sleep/wake states was done using the EEG and video recordings. B, Diagram of electrode layout. C, Magnification of EEG segments showing frontal delta and spindles (slow wave sleep) or 6–7 Hz rhythmic activity (wakefulness). EEG was done using the Stellate EEG system (Montreal, CA) with a Lamont Pro‐36 amplifier, sampling rate of 2 kHz. EEG is shown here using low and high frequency filters of 1 and 70 Hz, respectively. Time and sensitivity scales are shown for each age group. Please note the lower sensitivity in the P18 EEG recordings due to the higher amplitude signal at this older age. The figure was provided by Aristea Galanopoulou.
Figure 4
Figure 4
Evolution of EEG activity during early development using a wired recording system. The 4 representative epochs show EEG activity from cortex for each of 4 days from postnatal day 7 (P7) to P10 recorded from Sprague‐Dawley rats. The amplitude of the electrical activity increased with increasing age, from P7 to P10. Three behavioral states of the rat pup were observed during 3 corresponding EEG patterns for each epoch (P7 to P10). For each age (P7–P10, respectively), the EEG during the 3 different behaviors is shown below at an expanded time scale (AC): A shows the EEG activity during the awake state, which involved crawling, stretching, and yawning; B illustrates the EEG when no movement was observed, except for limb twitches and whole body jerks; and C shows the EEG when no movement was detected. The 3 EEG patterns in this figure, corresponding to the observed behaviors, are similar to those reported earlier by Jouvet‐Mounier et al. (1969), who recorded EEG from frontal cortex in P7 to P26 rats. These authors described the EEG corresponding to the awake state of the rat (A), to the early state of paradoxical sleep (B), and to quiet sleep (C). The EEG was recorded using a tethered system (Stellate Harmonie system, Natus Medical, San Carlos, CA, U.S.A.) with a silver wire (0.008 inch outer diameter) placed 2.5 mm behind the bregma and 3 mm lateral from midline sutures that was referenced to an electrode positioned near lambda in the same hemisphere. The electrode was placed just inside the cortex to obtain a good‐quality signal. The EEG data were collected with a sampling rate of 1,000 Hz, the default gain of 4,000×, and filters set at 0.5 Hz (low) and 70 Hz (high). The representative epochs of EEG shown in the figure were digitally filtered at 3.0 Hz (low) and 35 Hz (high) after the acquisition. The offline filters were applied to improve the visibility of amplitude differences between behavioral stages and across the ages. The figure is provided by Yogendra Raol.
Figure 5
Figure 5
Age‐dependent changes in background frequency bands in an awake freely behaving control rat pup. Each day between P7 and P11, background EEG was recorded from a set of 10 rat pups (A). For each of the EEG bands, power spectral density (PSD) in the EEG was estimated and the mean values were plotted with 95% confidence intervals (B). A substantive increase in power was observed between P7 and P8 across all frequency bands. Power in the beta and gamma bands progressively increased with age and showed a plateau at P10 and P11. Two recordings were conducted at P7 to verify stability and to evaluate the same‐day variability of the signal. Measurements of integrated power were compared with analysis of variance (ANOVA) (C). An asterisk shows statistical significance. Recordings were performed as described in Figure 1. The bandpass for the wireless recordings was 0.1–120 Hz. (For methods, please see methods in Ref. 30). Reprinted with permission from Zayachkivsky et al.30

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