The electroencephalogram (EEG) reflects brain electrical activity. A flat (isoelectric) EEG, which is usually recorded during very deep coma, is considered to be a turning point between a living brain and a deceased brain. Therefore the isoelectric EEG constitutes, together with evidence of irreversible structural brain damage, one of the criteria for the assessment of brain death. In this study we use EEG recordings for humans on the one hand, and on the other hand double simultaneous intracellular recordings in the cortex and hippocampus, combined with EEG, in cats. They serve to demonstrate that a novel brain phenomenon is observable in both humans and animals during coma that is deeper than the one reflected by the isoelectric EEG, and that this state is characterized by brain activity generated within the hippocampal formation. This new state was induced either by medication applied to postanoxic coma (in human) or by application of high doses of anesthesia (isoflurane in animals) leading to an EEG activity of quasi-rhythmic sharp waves which henceforth we propose to call ν-complexes (Nu-complexes). Using simultaneous intracellular recordings in vivo in the cortex and hippocampus (especially in the CA3 region) we demonstrate that ν-complexes arise in the hippocampus and are subsequently transmitted to the cortex. The genesis of a hippocampal ν-complex depends upon another hippocampal activity, known as ripple activity, which is not overtly detectable at the cortical level. Based on our observations, we propose a scenario of how self-oscillations in hippocampal neurons can lead to a whole brain phenomenon during coma.

Copyright: © 2013 Kroeger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The results presented here challenge the common wisdom that the isoelectric line is always associated with absent cerebral activity, and demonstrate that the isoelectric line is not necessarily one of the ultimate signs of a dying brain. We show that if cerebral neurons survive through the deepening of coma, then network activity can revive during deeper coma than the one accompanying the EEG isoelectric line by the change in the balance of hippocampal-neocortical interactions.

The activity of subcortical neurons (e.g. thalamic, hippocampal) has not been studied during EEG isoelectric line, but it might be hypothesized both from the situation encountered during the suppression phase of the BS pattern [5] and from recordings in isolated preparations (such as in vitro), that a rudiment of oscillatory activity might persist in subcortical neurons. Whether this activity can become synchronized and re-emerge at the cortical level is so far unknown.

The common clinical correlate of coma is loss of consciousness and low or absent responsiveness [4] . Although the initial stages (I–II) of coma are comparable to deep sleep [7] , deep coma (stages III-IV) corresponds to more profound alterations of brain states, observable at the electroencephalographic (EEG) level [8] , [9] . Deepening of the coma beyond the BS stage leads to a flat EEG called isoelectric line, which is presumed to be associated with silenced activity in cortical neurons. Such an EEG pattern is considered to be one of the limit points in establishing brain death and in particular clinical conditions it is accepted as the only criterion [10] .

BS was first described by Swank and Watson [3] . Its main feature at the EEG level consists of quasi-periodical bursts of bilateral high-amplitude slow waves (mainly <15 Hz) separated by low-amplitude or absent activity lasting from a few seconds to minutes [4] . The first cellular correlates of BS were revealed by Steriade and colleagues [5] , demonstrating that EEG bursts were associated with excitatory activities in cortical neurons, while suppression phases were paralleled by absence of cortical network interactions. The same study showed that thalamic neurons displayed spontaneous discharges throughout BS with no apparent correlation to the two phases of BS. Overall, BS was mainly investigated as a prognostic tool during coma (reviewed in [6] ).

Regardless of the underlying causes, coma is a state during which the brain reaches a low level of neuronal activity and metabolism. Possible etiologies range from safe and fully reversible pharmacological interventions (such as general anesthesia) to severe, irreversible brain damage. There is virtually no systematic investigation of the cerebral cellular mechanisms at work during coma and attempts to compare pathological and pharmacological etiologies are scarce. It is therefore no surprise that the outcome from coma is often predicted on a statistical basis. Recent results from our laboratory have highlighted two unexpected findings. First, we have shown that coma induced by a variety of anesthetics presents a time-frame during which the cortex is in a hyperexcitable state that is responsible for the genesis of the burst-suppression (BS) pattern [1] . Second, we reported evidence that isoflurane-induced BS opens the blood-brain barrier [2] .

The analysis was performed with software from WaveMetrics Inc. (Igor software, Oregon, USA), and relies on time relationships between the recorded voltage time series. Most of the results are presented as event- or spike-triggered averages. They were obtained after extracting from the intracellular or extracellular field/EEG potentials sweeps synchronized with the triggering event (action potentials or extreme of a particular waveform) and averaging them. This provided the statistical evidence for interactions between the discharge in the reference channel and the field/EEG potential variations of the target channel. Cross-correlograms were derived in order to detect time relationships between events of similar origin recorded from different channels. The procedure consisted in detecting events in two simultaneously recorded channels. The events in one channel were kept as time reference (time zero), and the time lags of the events in the other channel with respect to each event in the reference channel were plotted in a histogram.

Human EEG recordings were performed in monopolar configuration with the reference placed on the earlobes and crosslinked. Due to the supine position of the patient occipital electrodes could not be placed in the 10–20 classical configuration. Instead they were positioned more laterally, closer to the temporal ones. Filtering was set for all channels between 0.1 and 300 Hz, with the notch filter on.

Experiments were performed on twenty-six cats (2.5–4.5 kg) of both sexes. The surgical procedures were described in detail elsewhere [1] . After the initial dose of ketamine-xylazine (15 mg/kg and 3 mg/kg, respectively), animals were paralyzed (gallamine triethiodide), and anesthesia was switched to isoflurane (1.3–1.5%). After a stable baseline recording containing continuous slow and ample EEG waves, the isoflurane was increased to 4% in order to induce νC patterns. During the experiments vital parameters were continuously monitored and maintained within physiological limits: body temperature (37±0.2°C), expired CO 2 (3.7±0.2%), respiration rate (20–30 strokes/min) and heart rate (<110 beats/min). Through craniotomy we exposed the suprasylvian gyrus, where intracellular pipettes and field electrodes were lowered into the cortex and in the hippocampus using stereotaxic coordinates. The hippocampal pipettes were lowered through the suprasylvian gyrus and lateral ventricles aiming at the CA3 region of the hippocampus (AP+5; L+5.5; H+7.5 - in parallel to the midline). Neuroanatomical evidence for the placement of the recording electrodes was obtained for a few cells by staining neurons with intracellular injection of Lucifer yellow (3%, Invitrogen; dissolved in a 1 M LiCl 2 solution, Sigma). The intracellular filling was achieved by applying hyperpolarizing pulses (500 ms at 1 Hz) of 1–3 nA for 10–15 min. At the end of the experiments the brains were perfused with 4% paraformaldehyde and stored in paraformaldehyde and 30% sucrose for 2–3 days. Sections of 75 µm were taken and cells were revealed using a Zeiss confocal laser-scanning microscope (LSM 5 PASCAL, Zeiss, Oberkochen, Germany).

All experimental procedures were performed according to NIH guiding principles and were also approved by the committee for animal care of the university (Comité pour la protection des animaux de l′Université Laval, CPAUL). As for the human recording, written consent was obtained from the family in order to use the recordings performed during his treatment for publication and no experimental paradigm was implemented on the patient. The Committee for Ethics of the Unirea (Regina Maria) Medical Centre approved of the use of the recordings for publishing purposes.

Results

EEG Phenomena in Comatose Patients One of the main challenges in clinical practice is the fact that the interpretation of the EEG of a comatose patient is often hindered by the lack of information concerning the dynamic progression of the patient’s state. Such an exceptional case was represented by the EEG pattern displayed in Fig. 1. The patient was admitted in the emergency service after cardiorespiratory arrest and successful resuscitation. By the time of his arrival at the hospital he was unconscious and presented quasiperiodic generalized convulsions. He was administered antiepileptic medication (carbamazepine 3 times 200 mg per day, diazepam 3 times 10 mg per day, and thiopental i.v. 175 µg/kg/h) and this reduced the convulsions to quasiperiodic jerks. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. EEG recording of a patient in deep coma. (A1) Typical pattern of EEG recorded from temporal (T3 and T4) and modified occipital (O1′ and O2′) derivations. The pattern is dominated by ripples (Rs; bracket lines) and ν-complexes (νCs; oblique arrows). The recording also contains cardiac contamination, as often in comatose patients. (A2) Averaged νCs (n = 350). The negative peak of νCs in the O1′ derivation was detected and sweeps of 4 s around that time marker were extracted from each derivation and averaged. (A3) Averaged ripples (n = 5000). Left panel: Positive peak detection of Rs in the T4 derivation and averaging of all signals reveals no potentials in the occipital leads (not shown) and a reverted, less ample, ripple in T3. Right panel: similarly, positive peak detection in the O1′ derivation and averaging of all signals shows only a small and reverted ripple in O2′. (A4) Cross-correlogram between ripples in T4 and O1′ shows no time relationship. (B) Same patient, after removing antiepileptic drugs (carbamazepine, diazepam and thiopental) displays a pattern of burst-suppression. Here again, contamination of the EEG by the electrocardiogram, visible especially in the temporal leads. In this and following figures, all potentials are depicted with positivity upwards. https://doi.org/10.1371/journal.pone.0075257.g001 EEG recordings performed at this time showed a pattern like the one depicted in Fig. 1A1. Most unusually, it was dominated by bursts of rhythmic spike-like discharges (average frequency of 5.8±0.9 Hz; values are given as mean ± standard deviation throughout the paper), somewhat akin in shape and frequency to hippocampal ripple events (Rs), with each burst lasting for several seconds. They seemed to originate independently in several foci. We here give the example of those recorded in the T4 derivation (Fig. 1A3, left panel) and those of O1′ (Fig. 1A3, right panel). Each trace represents the average of 5000 ripple events. Ripples originating at one location were also recorded by contralateral electrodes with reversed polarity. The simultaneity of the two waveforms and their opposed polarity has been encountered in situations with an interposed dipole, although a definite conclusion is difficult to draw at this point. Moreover, there was no time relationship between ripples generated at different recording sites (Fig. 1A4) indicating that these events were not propagating through cerebral circuits but rather being generated by independent oscillatory structures and recorded by our electrodes through volume transmission. Other peculiar waveforms present during these recordings were ample and slower deflections occurring simultaneous with the motor jerks of the patient (indicated by oblique arrows in Fig. 1A1). For reasons that will become obvious further, we call this waveform ν-complex (νC). Its aspect is further detailed in Fig. 1A2 where averaged νCs (n = 350) are characterized by consistent polarity in any of the recorded leads (positive on the right, negative on the left). νCs appeared pseudo-rhythmically every ∼10 s (average time interval of 9.3±2.7 s; n = 350). After 6 days spent in this state, the antiepileptic medication was discontinued and, after passing through a period of isoelectric activity, the EEG assumed a pattern of burst-suppression (Fig. 1B) with frequent stereotyped burst shapes as described elsewhere [1]. This latter pattern is usually present during deep coma [4], and in this case was achieved by removing pharmacological agents such as thiopental reputed to deepen coma. Therefore we submit that under antiepileptic medication the patient was in a deeper state of coma. Moreover this state was accompanied by novel EEG patterns that are not described in the literature. We therefore planned a series of experiments with laboratory animals in order to elucidate the cellular mechanisms underlying this behavior.

EEG Phenomena in Comatose Animals Fig. 2A–D summarizes the essential EEG features accompanying the deepening of coma, as obtained with progressive doses of the anesthetic isoflurane applied in cats. Wakefulness (Fig. 2A) and sleep-like oscillations (Fig. 2B) have been extensively studied and their cellular and ionic substrates are well understood (for review, see [11]). Briefly, the EEG of wakefulness displays a relatively low amplitude and contains fast waves with a frequency spectrum generally above 15 Hz. Slow-wave sleep (SWS)-like patterns were induced with an initial dose of isoflurane of 1% (first blue arrow in Fig. 2). The EEG pattern of SWS is dominated by slow oscillations (<1 Hz) [12] intermingled with other rhythms such as delta (<4 Hz) and spindles (7–15 Hz) [11]. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. EEG patterns during wakefulness and diverse degrees of loss of consciousness. Cat EEG recording during application of various concentrations of isoflurane. Whilst the top-most panel depicts the complete sequence with arrows indicating the applications of increasing anesthesia, the underlined epochs are expanded below. (A) Undrugged preparation displaying low amplitude, fast (mostly >15 Hz) EEG. (B) Slow-wave sleep-like (SWS) pattern after 1% isoflurane, characterized by higher amplitude slow waves dominated by delta oscillations (<4 Hz). (C) Burst-suppression (BS) induced with 2% isoflurane showing alternating sequences of isoelectric line and bursting episodes. The latter are very similar to SWS patterns (see detail at right). (D) A further increase in the isoflurane concentration (3%) establishes a stable isoelectric line portraying the absence of phasic events. Only very low-amplitude activities can be observed at high gain. (E) Isoflurane at 4% elicits a revival of quasi-rhythmic spiky potentials of high amplitude, which we propose to call ν-complexes (or Nu-complexes; νC). https://doi.org/10.1371/journal.pone.0075257.g002 The BS pattern shown in Fig. 2C, first described by Swank and Watson [3], is associated with deep coma and generally develops during anesthesia (in our case, isoflurane at 2% applied at the 2nd arrow in Fig. 2), hypoxia, cardiac arrest, drug-related intoxications, childhood encephalopaties, hypothermia, etc. [13], [14], [15], [16], [17], [18]. The neurophysiological mechanisms responsible for generating BS patterns remained elusive for many years. Recently we have shown that BS induced with various anesthetics (isoflurane, propofol, barbiturates) is associated with a state of cortical hyperexcitability in that the bursts of the BS pattern can be triggered by subliminal stimuli reaching the hyperexcitable cortex [1]. Fig. 2D depicts the EEG isoelectric line following increased doses of isoflurane anesthesia (3% at the 3rd arrow in Fig. 2), reflecting a further deepening of the coma. Such EEG patterns are considered to portray “electrocerebral silence” and are one of the hallmarks in establishing brain death. Therefore, in clinical conditions, the presence of a prolonged isoelectric line in a comatose patient is, among others, one of the determinants of a fatal diagnosis [10]. The novelty reported in this study is that through an application of anesthetics beyond what is required for the induction of the isoelectric line (isoflurane >3.5%) we obtained a re-vitalization of activity in the brain, reflected by the EEG (Fig. 2E). This activity was characterized by sharp EEG waves occurring quasi-rhythmically at frequencies below 1 Hz. Since this EEG wave has been unknown until now, we propose to call it ν-complex (Nu-complex, νC), in deference to EEG tradition and its resemblance to the Greek letter “ν”. Given that such activity patterns have not been reported before, essential questions as to the site of genesis and underlying cellular mechanisms need answering.

Hippocampal Activities – Recordings of ν-Complexes in Cats At the EEG level, νCs were characterized by amplitudes of 1.3 mV (±0.2, n = 70) and duration of 129 ms (±25). Since νCs were clearly expressed in the EEG we assumed that they would primarily reflect cortical activities. Furthermore, our field potential (FP) recordings performed in all cortical areas (primary as well as associative ones) also displayed high amplitude νCs (3.4±0.4 mV) (Fig. 3A, upper trace). These FP events consisted almost exclusively of negative potentials in deep cortical layers (Fig. 3B5, upper trace), reflecting massive and synchronous cellular excitation. It became apparent from these recordings that νC-activity shared features with exclusively hippocampal phenomena such as the sharp waves during resting wakefulness and slow-wave sleep [19], and slow intrinsic spikes under urethane anesthesia [20]. Additional similarities were observed between νCs and less hippocampal-specific patterns such as epileptic interictal spikes [21]. Whether or not νCs were also present in the hippocampus or other subcortical structures was investigated with double field potential recordings. Virtually all tested structures (thalamus, basal forebrain, brainstem and hippocampus) displayed activities time-related with EEG νCs (not shown). However, among all subcortical structures, hippocampal νCs exhibited by far the greatest amplitude. Moreover, νCs from all other structures displayed a time-lag trailing hippocampal νC events by at least 10 ms, suggesting that the hippocampus might be the key structure for generating νCs. In more detail, measuring the time lags between the peaks of νCs recorded as field potentials we obtained the following values: basal forebrain was delayed with respect to the hippocampus by 14.2±3.7 ms (n = 240 νCs, data pooled from 3 animals), brainstem was delayed by 18.4±5.1 ms (n = 240, 3 animals), and thalamus (lateral pulvinar nucleus) was delayed by 9.8±4.6 ms (n = 240, from 3 animals). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. Isoflurane induction of deep coma, including the EEG isoelectric line and νC state. (A) Continuous recordings of intracortical field potentials (FP Cx) together with hippocampal field potentials (FP Hipp) first under lower levels of isoflurane (2%), which is then switched to 4% (at arrow) leading to a gradual suppression of bursting activity and eventually to isoelectric line. The 5 periods underlined by horizontal bars are expanded in (B). (B1) The magnification shows ripple activity in the hippocampus at ∼15 Hz during transient suppressions. (B2) Isolated burst surrounded by ripple activities in the hippocampus (∼9 Hz). (B3) & (B4) Continuous isoelectric line displaying progressively slower and ampler ripples in the hippocampal field potential. (B5) νC state characterized by ample delta ripples in the hippocampal trace interrupted occasionally by high amplitude νCs. Note that in all insets the overt expression of ripple activities in the cortex is absent. https://doi.org/10.1371/journal.pone.0075257.g003 Our simultaneous cortical and hippocampal field potential recordings also emphasized another activity exclusively present in this subcortical structure: faster ripple oscillations that were observable even during the cortical isoelectric line (Fig. 3). Indeed, these ripple oscillations were a constant feature starting with the state of BS (Fig. 3B1) and continuing throughout the transition to the EEG isoelectric line (Fig. 3B2–4) and νC state (Fig. 3B5). In all recordings of progressions from BS to νC state (n = 14) hippocampal ripple frequency slowed down continuously starting in the low beta range (15.8±0·9 Hz) during BS and ending within the delta range (2.2±0·7 Hz). In parallel with the slowing of the oscillatory frequency we noted an increase in the amplitude of the ripples by an average factor of 10, suggesting a progressive increase in the synchronization within hippocampal networks during ripple activity. Interestingly, νCs appeared to reset the amplitude scale for ripples (Fig. 3B5) in that the first event after a νC was dramatically smaller, whilst subsequent ripples displayed successively larger amplitudes. Although ripple activity was not overtly visible in cortical recordings we noticed that at higher amplifications and with the benefit of simultaneous hippocampal field potentials, one could distinguish coherent phasic potentials even in the cortical field potential (Fig. 3B3–5). This observation raised the question as to whether such FP events portray true intracellular oscillations by cortical neurons, or are merely the reflection of hippocampal dipoles.