Florin Amzica

Electrophysiological correlates of sleep delta waves.

Recent studies have disclosed several oscillations occurring during resting sleep within the frequency range of the classical delta band (0.5-4 Hz). There are at least 3 oscillations with distinct mechanisms and sites of origin: a slow (<1 Hz) cortically-generated oscillation, a clock-like thalamic oscillation (1-4 Hz), and a cortical oscillation (1-4 Hz). The present paper reviews data on these oscillations and the possible mechanisms which coalesce them into the polymorphic waves of slow wave sleep. Data stem from intracellular (over 500 single cell and 50 double impalements) and field potentials recorded from the cortex and thalamus of cats (120 animals) under ketamine and xylazine anesthesia. Other experiments were based on whole night EEG recordings from humans (5 subjects). The frequency of the slow oscillation both in anesthetized animals and in naturally sleeping humans ranged between 0.1 and 1 Hz (89% of the cases being between 0.5 and 0.9 Hz). The slow (<1 Hz) oscillation is reflected in the EEG as rhythmic sequences of surface-negative waves (associated with hyperpolarizations of deeply-lying neurons) and surface-positive K-complexes (representing excitation in large pools of cortical neurons). Through its long-range synchronization, the slow oscillation has the ability to trigger and to group thalamically-generated spindles and two delta (1-4 Hz) oscillations. Finally, it is argued that the analysis of the electroencephalogram should transcend the spectral analyses, by taking into account the shape of the waves and, when possible, the basic mechanisms that generate those waves.

The K-complex: Its slow (<1-Hz) rhythmicity and relation to delta waves

The K-complex is a major graphoelement of sleep EEG. This report demonstrates that K-complexes emerge from a cortically generated slow(≤1-Hz) oscillation. Human EEG as well as cat cellular and field potential recordings converge into demonstrating that the K-complex results from a synchronized cortical network that imposes periodic excitatory and inhibitory actions on cortical neurons. We additionally show the correspondence between neuronal activities and the shape of the K-complex. Spectral analysis confirms the periodic recurrence of human K-complexes, with main peaks at 0.5 to 0.7 Hz. It is also shown that the spectral content in the delta band (1 to 4 Hz) is partially due to the shape and duration of the K-complex.

Synchronized sleep oscillations and their paroxysmal developments

The state of resting sleep is associated with a series of oscillations generated in cortical and thalamic networks. A newly discovered rhythm groups the spindle and delta sleep oscillations within slowly recurring (< 1 Hz) sequences. Multi-site, extra- and intracellular recordings provide evidence for synchronization of various classes of cell in the neocortex and thalamus during sleep oscillations that might reach paroxysmal levels similar to epileptic states. Sleep oscillations and the underlying synchronizing processes are disrupted during transition to brain arousal.

Cortical and thalamic cellular correlates of electroencephalographic burst-suppression.

This experimental study on anesthetized cats used intracellular recordings of cortical, thalamocortical and reticular thalamic neurons (n = 54), as well as multi-site extracellular recordings (n = 36), to investigate the cellular correlates of EEG burst-suppression patterns, defined as alternating wave bursts and periods of electrical silence. Burst-suppression was elicited by the administration of the same or other anesthetic agents upon the background of an already synchronized EEG activity. About 95% of cortical cells entered burst-suppression, in close time-relation with EEG activity, displaying sequences of phasic depolarizing events associated with bursts of EEG waves and an electrical silence of the neuronal membrane during flat EEG epochs. The membrane potential (Vm) hyperpolarized by approximately 10 mV prior to any EEG change and the slow rhythms reflecting deep stages of anesthesia progressively disorganized with transition to burst-suppression. During flat EEG epochs, the apparent input resistance (tested through short hyperpolarizing current pulses) decreased (range 12-60%) and neuronal responsiveness to orthodromic volleys (tested by thalamic and cortical evoked excitatory postsynaptic potentials) was dramatically reduced. It is proposed that the decreased input resistance is mainly due to an increase in K+ conductances. At variance with cortical neurons, only 60-70% of thalamic cells ceased firing before overt EEG burst-suppression and were completely silent during flat periods of EEG activity. The remaining 30-40% of thalamic cells discharged rhythmic (1-4 Hz) spike bursts during periods of EEG silence. This rhythm, within the frequency range of delta waves, is generated in thalamic cells by the interplay between two of their intrinsic currents at critical levels of Vm hyperpolarization. However, with the deepening of burst-suppression, when silent EEG periods became longer than 30 sec, thalamic cells also ceased firing. The assumption that full-blown burst-suppression is achieved through virtually complete disconnection in brain circuits implicated in the genesis of the EEG is corroborated by the revival of normal cellular and EEG activities after volleys setting into action thalamic and cortical networks.

Cellular substrates and laminar profile of sleep K-complex.

We describe the cellular mechanisms that underlie the generation of the K-complex, a major grapho-element of sleep electroencephalogram in humans. First we demonstrate the similarity between K-complexes recorded during natural sleep and under ketamine-xylazine anaesthesia in cats. Thereafter, we show by means of multi-site cellular and field potential recordings that K-complexes are rhythmic at frequencies of less than 1 Hz (mainly 0.5-0.9 Hz) and that they are synchronously distributed over the whole cortical surface as well as transferred to the thalamus. The surface K-complex reverses its polarity at a cortical depth of about 0.3 mm. At the cortical depth, the K-complex is made of a sharp and high-amplitude negative deflection that reflects cellular depolarization, often preceded by a smaller-amplitude, positive slow-wave reflecting cellular hyperpolarization. The sharp component of the K-complex may lead to a spindle sequence and/or to fast (mainly 20-50 Hz) oscillations. K-complexes appear spontaneously or triggered by cortical or thalamic stimulation, and they arise within cortical networks. We suggest that K-complexes, either in isolation or followed by a brief sequence of spindle waves, are the expression of the spontaneously occurring, cortically generated slow oscillation.

Implication of Kir4.1 Channel in Excess Potassium Clearance: An In Vivo Study on Anesthetized Glial-Conditional Kir4.1 Knock-Out Mice

The Kir4.1 channel is crucial for the maintenance of the resting membrane potential of glial cells, and it is believed to play a main role in the homeostasis of extracellular potassium. To understand its importance in these two phenomena, we have measured in vivo the variations of extracellular potassium concentration ([K+]o) (with potassium-sensitive microelectrodes) and membrane potential of glial cells (with sharp electrodes) during stimulations in wild-type (WT) mice and glial-conditional knock-out (cKO) Kir4.1 mice. The conditional knockout was driven by the human glial fibrillary acidic protein promoter, gfa2. Experiments were performed in the hippocampus of anesthetized mice (postnatal days 17–24). Low level stimulation (<20 stimuli, 10 Hz) induced a moderated increase of [K+]o (<2 mm increase) in both WT and cKO mice. However, cKO mice exhibited slower recovery of [K+]o levels. With long-lasting stimulation (300 stimuli, 10 Hz), [K+]o in WT and cKO mice displayed characteristic ceiling level (>2 mm increase) and recovery undershoot, with a more pronounced and prolonged undershoot in cKO mice. In addition, cKO glial cells were more depolarized, and, in contrast to those from WT mice, their membrane potential did not follow the stimulation-induced [K+]o changes, reflecting the loss of their high potassium permeability. Our in vivo results support the role of Kir4.1 in setting the membrane potential of glial cells and its contribution to the glial potassium permeability. In addition, our data confirm the necessity of the Kir4.1 channel for an efficient uptake of K+ by glial cells.

Physiology of sleep and wakefulness as it relates to the physiology of epilepsy.

This paper reviews the present knowledge about the cellular origins of vigilance states (wakefulness and slow-wave sleep) from the perspective of their involvement in the triggering of epileptic seizures. The data stem from intracellular recordings (most of them dual impalements of pairs of neurons and glia), extracellular ionic concentrations (mainly K+ and Ca2+) and simultaneous intracortical field potentials from the cortex of cats. These data were corroborated with recordings from naturally sleeping animals and humans. It is shown that sleep is dominated by a cortically generated slow (<1 Hz) oscillation resulting from the complex interplay within networks of neurons and glia, which are modulated by the more diffuse action of extracellular currents of ions. Wakefulness is produced through the activation of brainstem and basal forebrain structures, which disrupt sleep oscillations and elicit a global change of the extraneuronal milieu, with profound modifications of glial and cerebral blood flow parameters. Paroxysmal events arising during quiet sleep evolve within the cortex from normal slow sleep oscillations. The synchronization of large cortical and eventually subcortical territories relies on the propagation of increased currents of K+ through the glial syncytium, which compensate for the reduced synaptic efficacy due to the depletion of extracellular Ca2+.

Voltage-dependent fast (20-40 Hz) oscillations in long-axoned neocortical neurons.

Fast (20-80 Hz) oscillations of cortical activity, occurring during an increased level of focused alertness or elicited by optimal sensory stimuli, have been described by recording field potentials and neuronal firing in various cortical areas. Despite the increasing interest in this topic, little is known about the cellular mechanisms of the fast (generally termed 40-Hz) rhythm. An in vitro study demonstrated that, in sparsely spiny interneurons of frontal cortex, the 40-Hz rhythm is generated by a voltage-dependent persistent Na+ current, with the involvement of a delayed rectifier. Here we report depolarization-dependent 40-Hz oscillations in cats motor and association neocortical neurons with identified projections to contralateral homotopic cortical area and thalamus. Our data indicate that this fast rhythm may be synchronized through intracortical and corticothalamic linkages.

Opening of the blood–brain barrier during isoflurane anaesthesia

In order to produce its desired effect, anaesthesia acts upon neuronal elements by modifying membrane conductances and transmitter interactions. The effect of higher doses of isoflurane, widely used in clinical settings, on the permeability of the blood–brain barrier (BBB) is meanwhile ignored. In this study we investigated the integrity of the BBB during various levels of isoflurane anaesthesia (1% and 3%) in cats by monitoring the extravasation of Evans blue. Simultaneously we measured the electroencephalogram (EEG), with particular emphasis on its direct current (DC) component. High doses of anaesthetic (3%) broke down the BBB in the cortex and thalamus, while milder doses (1%) only opened the BBB in the thalamus. The fluorescent signal of Evans blue was visible over an extravascular length of 23 μm in the cortex and 25 μm in the thalamus, similar to the diffusion of the same dye when the BBB was disrupted with mannitol. The opening of the BBB was associated with (i) a positive DC shift in the EEG measured on the scalp and (ii) an evaluated increase in cerebral volume of 2–2.8%. The opening of the BBB by high doses of isoflurane brings into discussion hitherto unexplored effects of anaesthesia on the brain. The electrophysiological correlate provided by the DC component of the EEG constitutes a promising option for the assessment of the BBB integrity during human anaesthesia.

Basic physiology of burst-suppression

Burst-suppression (BS) is an electroencephalography (EEG) pattern consisting of alternative periods of slow waves of high amplitude (the burst) and periods of socalled flat EEG (the suppression) (Swank & Watson, 1949). It is generally associated with comatose states of various etiologies (hypoxia, drug-related intoxication, hypothermia, and childhood encephalopathies, but also anesthesia). It has been studied extensively at the EEG level (see review by Brenner, 1985, also this issue), but only sparse information is available with respect to the cellular and ionic mechanisms underlying its patterns. Some of the most fascinating questions pertain to the genesis of bursts: Are they truly spontaneous, what triggers them, what mechanism dictates their quasi-periodicity? Moreover, in clinical practice bursting activities during BS are often associated with jerks resembling those present during epileptic fits. Is there any common link to known seizure mechanisms? At the cortical level, EEG bursts are always associated with phasic synaptic depolarizing intracellular potentials, occasionally crowned by action potentials, in virtually all recorded cortical neurons (Steriade et al., 1994; see Fig. 1A, left panel). This study has also shown that suppression episodes are due to absence of synaptic activity among cortical neurons. However, it was also shown that some thalamocortical neurons display a rhythmic activity in the frequency range of delta oscillations (1–4 Hz) during suppressed periods. Recently we have shown further that BS represents a distinct behavioral frame during which the cortical network is in a hyperexcitable state, and that bursting activity may be triggered by subliminal stimuli (Kroeger & Amzica, 2007; Fig. 1). The cortical hyperexcitability was demonstrated under various anesthetics ranging from those enhancing Cl inhibition (propofol, barbiturates) to those boosting glutamate uptake (isoflurane). In the latter case, hyperexcitability resulted from the reduction of cortical inhibition (Ferron et al., 2009), which was corroborated with an outburst of extracellular Cl, probably reflecting the lesser activity of c-aminobutyric acid (GABA)A inhibitory synapses. It results that the excitatory–inhibitory balance leans toward excitation. The bursting process is limited in time because bursting activity is accompanied by a depletion of extracellular cortical Ca at levels that are incompatible with synaptic transmission. This generates an overall disfacilitation in cortical networks (Kroeger & Amzica, 2007), which ultimately is responsible for the arrest of neocortical neuronal activities and the ensuing flat EEG. During suppression, the synaptic silence allows neuronal pumps to restore interstitial Ca levels at control levels. At this moment, any external (or intrinsic) signal is able to trigger a new burst in the hyperexcitable cortex. Therefore, the pseudo-rhythmicity of the BS pattern is dictated by the degree of extracellular Ca depletion and the ability of neurons to restore this concentration. These phenomena are modulated by the general state of the nervous system and, therefore, the etiology and the seriousness of the condition. As coma deepens, bursting episodes become shorter, whereas the opposite happens to the suppression, leading eventually to continuous isoelectric EEG. The impaired ability of the central nervous system to keep extracellular Ca ions at normal levels might be precipitated by the fact that, at least as demonstrated with isoflurane, the permeability of the blood–brain barrier is compromised during BS (T trault et al., 2008). An interesting issue concerns the similarity between symptoms associated either with bursts during BS or with spike–wave seizures. Moreover, both conditions occur on a background of impaired inhibition. Furthermore, in clinical practice there is often unclear delimitation between comatose BS behavior and epileptic manifestations (e.g., in Hirsch et al., 2004). In addition, the antiepileptic medication obtains poor response (Dan & Boyd, 2006). This calls for one of the two possibilities: Either BS is included in the already complex syndrome of epilepsies (with complicating issues regarding mechanisms and curative strategies) or it is regarded as distinct processes with distinct Address correspondence to Florin Amzica, Department of Stomatology, School of Dentistry, Universit de Montreal, C.P. 6128, succursale Centre-ville, Montreal H3C 3J7, QC, Canada. E-mail: florin.amzica@ umontreal.ca