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Electrical Stimulation of the Anterior Cingulate Gyrus Induces Responses Similar to K-complexes in Awake Humans

  • Author Footnotes
    1 Drs Z. Voysey and D. Martín-López are joint first authors.
    Zanna Voysey
    Footnotes
    1 Drs Z. Voysey and D. Martín-López are joint first authors.
    Affiliations
    Department of Basic and Clinical Neuroscience, King's College London, Institute of Psychiatry, Psychology and Neuroscience, UK
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  • Author Footnotes
    1 Drs Z. Voysey and D. Martín-López are joint first authors.
    David Martín-López
    Footnotes
    1 Drs Z. Voysey and D. Martín-López are joint first authors.
    Affiliations
    Department of Basic and Clinical Neuroscience, King's College London, Institute of Psychiatry, Psychology and Neuroscience, UK

    Department of Clinical Neurophysiology, King's College Hospital NHS Trust, London, UK

    West Surrey Clinical Neurophysiology, St Peter's Hospital, Chertsey, UK
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  • Diego Jiménez-Jiménez
    Affiliations
    Department of Basic and Clinical Neuroscience, King's College London, Institute of Psychiatry, Psychology and Neuroscience, UK

    Department of Clinical Neurophysiology, King's College Hospital NHS Trust, London, UK

    Universidad San Francisco de Quito, School of Medicine, Quito, Ecuador
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  • Richard P. Selway
    Affiliations
    Department of Neurosurgery, King's College Hospital NHS Trust, London, UK
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  • Author Footnotes
    2 Drs G. Alarcón and A. Valentín are joint senior authors.
    Gonzalo Alarcón
    Footnotes
    2 Drs G. Alarcón and A. Valentín are joint senior authors.
    Affiliations
    Department of Basic and Clinical Neuroscience, King's College London, Institute of Psychiatry, Psychology and Neuroscience, UK

    Department of Clinical Neurophysiology, King's College Hospital NHS Trust, London, UK

    Departamento de Fisiología, Facultad de Medicina, Universidad Complutense, Madrid, Spain

    Neurology Section, Medicine Department, Hamad Medical Corporation, Doha, Qatar
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  • Author Footnotes
    2 Drs G. Alarcón and A. Valentín are joint senior authors.
    Antonio Valentín
    Correspondence
    Corresponding author. Department of Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, PO 43, De Crespigny Park, London SE5 8AF, UK. Tel.: +44 20 7848 5161x5436; fax: +44 20 73463725.
    Footnotes
    2 Drs G. Alarcón and A. Valentín are joint senior authors.
    Affiliations
    Department of Basic and Clinical Neuroscience, King's College London, Institute of Psychiatry, Psychology and Neuroscience, UK

    Department of Clinical Neurophysiology, King's College Hospital NHS Trust, London, UK

    Departamento de Fisiología, Facultad de Medicina, Universidad Complutense, Madrid, Spain
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  • Author Footnotes
    1 Drs Z. Voysey and D. Martín-López are joint first authors.
    2 Drs G. Alarcón and A. Valentín are joint senior authors.

      Highlights

      • SPES of the anterior cingulate gyrus induces EEG responses resembling K-complexes.
      • Responses similar to K-complexes can be induced during wakefulness.
      • This is the first causal evidence that the cingulate gyrus initiates K-complexes.

      Abstract

      Background

      The brain region responsible for the initiation of K-complexes has not been identified to date.

      Objective

      To determine the brain region responsible for originating K-complexes.

      Methods

      We reviewed all 269 patients assessed for epilepsy surgery with intracranial electrodes and single pulse electrical stimulation (SPES) at King's College Hospital between 1999 and 2013. Intracranial EEG responses to electrical stimulation at orbitofrontal, frontal, cingulate, temporal and parietal loci were compared visually with each patient's K-complexes and the degree of resemblance was quantified.

      Results

      Among the 269 patients, K-complex-like responses were exclusively observed in all 6 patients who had depth electrodes in the cingulate cortex. In each patient, the stimulation site eliciting the response of greatest similarity to the patient's K-complex was located within the dorso-caudal anterior cingulate. The K-complex like responses were evoked when the patients were awake.

      Conclusion

      Our findings provide the first causal evidence that the cingulate gyrus initiates the widespread synchronous activity that constitutes the K-complex. The induction of K-complex-like responses during wakefulness suggests that the mechanisms required for the initiation of K-complexes are separate from those involved in sleep.

      Keywords

      Abbreviations:

      EEG (Electroencephalography), SPES (Single pulse electrical stimulation), r (Correlation coefficient), SI (Similarity index)

      Introduction

      K-complexes are electroencephalographic (EEG) phenomena occurring during sleep, arising either spontaneously or in response to sensory stimulation. They are identified as a large biphasic negative-positive wave lasting for longer than 0.5 s, often preceded or followed by a sleep spindle [
      • Iber C.
      • Ancoli-Israel S.
      • Chesson A.L.
      • Quan S.F.
      AASM – Manual for the scoring of sleep and associated events.
      ].
      Since K-complexes were first described in 1938 by Loomis et al. [
      • Loomis A.L.
      • Harvey E.N.
      • Hobart G.A.
      Distribution of disturbance-patterns in the human electroencephalogram, with special reference to sleep.
      ], they have become a marker of stage II sleep. Their physiological mechanisms are largely unknown, with evidence supporting both a sleep-protective role [
      • Amzica F.
      • Steriade M.
      The functional significance of K-complexes.
      ], and conversely, one of arousal [
      • Halász P.
      • Pál I.
      • Rajna P.
      K-complex formation of the EEG in sleep. A survey and new examinations.
      ]. Likewise the axiom that they can arise spontaneously is under question, with some evidence to suggest that seemingly-spontaneous K-complexes may in fact be a response to unrecognized internal stimuli, such as borborygmi [
      • Johnson L.G.
      • Karpan W.E.
      Autonomic correlates of the spontaneous K-complex.
      ,
      • Niiyama Y.
      • Satoh N.
      • Kutsuzawa O.
      • Hishikawa Y.
      Electrophysiological evidence suggesting that sensory stimuli of unknown origin induce spontaneous K-complexes.
      ].
      Above all, however, it is their generation that remains controversial. Studies combining multisite cellular, local field and scalp EEG recordings [
      • Amzica F.
      • Steriade M.
      Short- and long-range neuronal synchronization of the slow (<1 Hz) cortical oscillation.
      ,
      • Cash S.S.
      • Halgren E.
      • Dehghani N.
      • et al.
      The human K-complex represents an isolated cortical down-state.
      ,
      • Contreras D.
      • Esteriade M.
      Cellular basis of EEG slow rhythms: corticothalamic relationships.
      ,
      • Steriade M.
      • Nuñez A.
      • Amzica F.
      A novel slow (<1 Hz) oscillation of neocortical neurons in vivo: depolarizing and hyperpolarizing components.
      ,
      • Steriade M.
      • Contreras D.
      • Curró Dossi R.
      • Nuñez A.
      The slow (<1 Hz) oscillation in reticular thalamic and thalamocortical neurons: scenario of sleep rhythm generation in interacting thalamic and neocortical networks.
      ,
      • Steriade M.
      • Nuñez A.
      • Amzica F.
      Intracellular analysis of relations between the slow (<1 Hz) neocortical oscillation and other sleep rhythms of the electroencephalogram.
      ] suggest that K-complexes arise from synchronized activity across widespread cortical areas mediated by hyperpolarising currents in layer III of the cortex, such that they form a transient version of the on-going slow wave activity that predominates in stage III–IV sleep [
      • Hirsch J.C.
      • Fourment A.
      • Marc M.E.
      Sleep-related variations of membrane potential in the lateral geniculate body relay neurons of the cat.
      ]. This activity then propagates via cortico-cortical connections [
      • Amzica F.
      • Steriade M.
      Short- and long-range neuronal synchronization of the slow (<1 Hz) cortical oscillation.
      ] in a predominantly anterior-posterior direction [
      • Nir Y.
      • Staba R.J.
      • Andrillon T.
      • et al.
      Regional slow waves and spindles in human sleep.
      ,
      • Massimini M.
      • Huber R.
      • Ferrarelli F.
      • Hill S.
      • Tononi G.
      The sleep slow oscillation as a traveling wave.
      ]. It follows, however, that there must be a region that orchestrates and synchronizes the initial hyperpolarising input to widespread cortical regions, the location of which remains unknown.
      The areas originating K-complexes are much debated. As long ago as the 1950s, the cingulate gyrus was proposed as the area originating K-complexes [
      • Roth M.
      • Shaw J.
      • Green J.
      The form voltage distribution and physiological significance of the K-complex.
      ,
      • Gastaut H.
      Etude électrographique chez l'homme et chez l'animal des décharges épileptiques dites psychomotrices.
      ]. Initially this was based on source-localization from scalp EEG recordings, but such conclusions were limited by poor spatial coverage and the tendency for traditional simple dipole models to attribute diffuse bilateral cortical activity to a deep midline source. More sophisticated contemporary source-localization techniques employing high definition EEG and distributed source modeling could not provide convincing evidence for the involvement of the cingulate gyrus in the generation of K-complexes [
      • Wennberg R.
      • Cheyne D.
      On noninvasive source imaging of the human K-complex.
      ,
      • Murphy M.
      • Riedner B.A.
      • Huber R.
      • Massimini M.
      • Ferrarelli F.
      • Tononi G.
      Source modeling sleep slow waves.
      ]. EEG-fMRI studies [
      • Dang-Vu T.T.
      • Desseilles M.
      • Laureys S.
      • et al.
      Cerebral correlates of delta waves during non-REM sleep revisited.
      ,
      • Czisch M.
      • Wehrle R.
      • Stiegler A.
      • et al.
      Acoustic oddball during NREM sleep: a combined EEG/fMRI study.
      ] have implicated deep midline structures including the cingulate gyrus in some aspects of K-complex physiology, but the area responsible for initiating K-complexes was not identified. Intracranial EEG has provided more direct insight into this question. Early studies in cats [
      • Hughes J.R.
      • Mazurowski J.A.
      Studies on the supracallosal mesial cortex of unanesthetized, conscious mammals: I. Cat. B. Electrical activity.
      ] and monkeys [
      • Hughes J.R.
      • Mazurowski J.A.
      Studies on the supracallosal mesial cortex of unanesthetized, conscious mammals: II. Monkey. D. vertex sharp waves and epileptiform activity.
      ] highlighted a cingulate generator for the K-complex, but the transferability of such findings to humans is questionable. In contrast, intracranial EEG recordings in humans have suggested that the cingulate cortex and functionally related mesial frontal structures appear uninvolved in generating the visible human K-complex waveform [
      • Wennberg R.
      Intracranial cortical localization of the human K-complex.
      ], although the site of initiation of the K-complex could not be resolved at the level of EEG macroelectrodes.
      At our center, we have developed a novel approach to investigate the generators of K-complexes. Patients studied with intracranial EEG recordings routinely undergo assessment with single pulse electrical stimulation (SPES) to aid identification of epileptogenic cortex [
      • Alarcón G.
      • Martinez J.
      • Kerai S.V.
      • et al.
      In vivo neuronal firing patterns during human epileptiform discharges replicated by electrical stimulation.
      ,
      • Flanagan D.
      • Valentín A.
      • García Seoane J.J.
      • Alarcón G.
      • Boyd S.G.
      Single-pulse electrical stimulation helps to identify epileptogenic cortex in children.
      ,
      • Valentín A.
      • Alarcón G.
      • Honavar M.
      • et al.
      Single pulse electrical stimulation for identification of structural abnormalities and prediction of seizure outcome after epilepsy surgery: a prospective study.
      ,
      • Valentín A.
      • Alarcón G.
      • García-Seoane J.J.
      • et al.
      Single-pulse electrical stimulation identifies epileptogenic frontal cortex in the human brain.
      ,
      • Valentín A.
      • Anderson M.
      • Alarcón G.
      • et al.
      Responses to single pulse electrical stimulation identify epileptogenesis in the human brain in vivo.
      ]. Essentially, a single small electrical pulse (3–5 mA amplitude, 1 ms duration) is applied between contiguous electrodes, and EEG responses are recorded by remaining electrodes. In the course of our practice, we observed that stimulation of some regions induced responses of marked similarity to the patient's K complexes. In the present work, we report these areas, and quantify the resemblance between SPES responses and K-complexes. To our knowledge, we provide the first causal evidence that K-complexes are initiated by the cingulate cortex.

      Methods

      Subjects

      Records from all 269 patients assessed for epilepsy surgery with intracranial electrodes and SPES at King's College Hospital (London) between January 1999 and December 2013 were reviewed. The study included all patients who had:
      • 1.
        Bihemispheric intracerebral (depth) electrodes.
      • 2.
        Frontal lobe intracerebral electrodes.
      • 3.
        Intracranial sleep stage II recordings.
      Patients with the following criteria were excluded:
      • 1.
        Previous resective neurosurgery.
      • 2.
        Continuous abnormalities in the background EEG precluding identification of K-complexes and/or SPES responses.
      The development of SPES was approved by the ethical committee of King's College Hospital (99–017). SPES is now part of the clinical protocol for presurgical assessment of patients with epilepsy with intracranial recordings.

      Electrodes

      In all patients, bilateral multicontact flexible bundles of depth (intracerebral) electrodes (AdTech Medical Instruments Corp., WI, USA) were implanted stereotactically under MRI guidance at sites which included frontal, parietal and temporal locations. Each electrode bundle contained 6–10 cylindrical 2.3 mm platinum electrodes, with adjacent electrode centers separated by 5 mm. The location of depth electrodes was verified by postimplantation skull X-ray, brain CT or MRI. The type, number and location of the electrodes were determined by the suspected location of the ictal onset region, according to non-invasive evaluation: clinical history, scalp EEG recordings obtained with the Maudsley system [
      • Kissani N.
      • Alarcon G.
      • Dad M.
      • Binnie C.D.
      • Polkey C.E.
      Sensitivity of recordings at sphenoidal electrode site for detecting seizure onset: evidence from scalp, superficial and deep foramen ovale recordings.
      ,
      • Fernández Torre J.L.
      • Alarcón G.
      • Binnie C.D.
      • et al.
      Generation of scalp discharges in temporal lobe epilepsy as suggested by intraoperative electrocorticographic recordings.
      ], neuropsychology [
      • Akanuma N.
      • Koutroumanidis M.
      • Adachi N.
      • Alarcón G.
      • Binnie C.D.
      Presurgical assessment of memory-related brain structures: the Wada test and functional neuroimaging.
      ] and neuroimaging. The selection criteria and implantation procedure have been described elsewhere [
      • Alarcón G.
      • Martinez J.
      • Kerai S.V.
      • et al.
      In vivo neuronal firing patterns during human epileptiform discharges replicated by electrical stimulation.
      ].

      Recording protocol

      Recording of intracranial EEG started when the patient had recovered from electrode implantation, usually 24–48 h after surgery. Cable telemetry with up to 64 recording channels was used for data acquisition with simultaneous video monitoring. In two patients, a Telefactor Beehive-Beekeeper system (Astro-Med, RI, USA) was used. Data were digitized at 200 Hz and band pass filtered (high pass cut-off frequency at 0.3 Hz and low pass cut-off frequency at 70 Hz). The system input range was 2 mV and data were digitized with a 12 bit analog-to-digital converter (amplitude resolution of 0.976 μV). In four patients, a Medelec-Profile system was used (Medelec, Oxford Instruments, United Kingdom). Data were digitized at 256 Hz (two patients) or 1024 Hz (two patients) and band pass filtered (0.05–70 Hz). The input range was 10 mV and data were digitized with a 22 bit analog-to-digital converter (an amplitude resolution of 0.153 μV). Interictal awake and sleep recordings in addition to ictal recordings were permanently stored in hard drives. Data were recorded as common reference to Cz-Pz or to an intracranial electrode, and displayed in a variety of montages, including common average reference.

      SPES protocol

      SPES was applied sequentially between pairs of adjacent electrodes with a constant current neurostimulator (Medelec ST10 Sensor, Oxford Instruments or Leadpoint, Medtronic) using monophasic single pulses (0.1–0.2 Hz, 1 ms, 3–5 mA). At least 20 stimuli were delivered at for each stimulated site. Either all electrodes (patients 1, 3, 5 and 6) or only the electrodes located in grey matter (patients 2 and 4) were used to stimulate. EEG responses to each pulse were recorded through the non-stimulating electrodes. A more detailed description of the experimental protocol for SPES is described elsewhere [
      • Flanagan D.
      • Valentín A.
      • García Seoane J.J.
      • Alarcón G.
      • Boyd S.G.
      Single-pulse electrical stimulation helps to identify epileptogenic cortex in children.
      ,
      • Valentín A.
      • Alarcón G.
      • Honavar M.
      • et al.
      Single pulse electrical stimulation for identification of structural abnormalities and prediction of seizure outcome after epilepsy surgery: a prospective study.
      ,
      • Valentín A.
      • Alarcón G.
      • García-Seoane J.J.
      • et al.
      Single-pulse electrical stimulation identifies epileptogenic frontal cortex in the human brain.
      ,
      • Valentín A.
      • Anderson M.
      • Alarcón G.
      • et al.
      Responses to single pulse electrical stimulation identify epileptogenesis in the human brain in vivo.
      ]. SPES protocol was always started with the patient awake. SPES responses resembling K-complexes will be called “K-complex like” responses to emphasize that, despite their resemblance to K-complexes, SPES responses did not fulfill the standard definition of K-complexes (occurrence during sleep either spontaneously or in response to sensory stimulation).

      Identification of K-complexes

      In contrast to scalp recordings, where K complexes are usually bilaterally symmetrical and largest at the midline, there are not established criteria to identify K-complexes in intracranial recordings. We have followed some of the standard scalp criteria in addition to those described by Wennberg [
      • Wennberg R.
      Intracranial cortical localization of the human K-complex.
      ] with simultaneous intracranial and scalp recordings. In essence, we identified K-complexes in our intracranial records as high amplitude (>50 μV) bilateral frontal biphasic waveforms lasting longer than 0.5 s, often preceded or followed by a sleep spindle. The presence of bilateral intracranial frontal electrodes was required in our patient population because our cohort included patients with focal frontal epilepsy, who may show unilateral sleep-activated focal discharges that could be wrongly identified as K complexes.

      Comparison of recordings

      Intracranial EEG was analyzed using ASA4.8.1™ in referential montage (reference to Cz or Pz). Researchers undertaking EEG analysis were blinded to electrode placement and patient details. The first 20 K-complexes occurring during sleep were identified visually, and 4 s epochs centered at the event were averaged to create a per-patient averaged K-complex. K-complex selection was reviewed independently by authors AV and GA. Interictal awake recordings were also reviewed for K-complex-like spontaneous interictal epileptiform discharges. Where present, the first 20 of such discharges were averaged using the same method. Similarly, for each pair of stimulating electrodes, 20 SPES responses were averaged in 4-s epochs and compared visually against the average of K-complexes or spontaneous interictal epileptiform discharges. For each electrode bundle, the averaged responses of greatest visual similarity to the averaged K-complex and/or epileptiform discharge were submitted for quantitative analysis as below.

      Quantitative analysis

      Computer software was implemented in Matlab (The Math Works Inc., USA) to quantify the similarity between averaged SPES responses, averaged K complexes and averaged spontaneous interictal epileptiform discharges.
      For each patient, averaged K-complexes, responses to SPES and spontaneous interictal epileptiform discharges (where available) were compared to each other in one-to-one comparisons. Because there was no a priori synchronizing marker for recordings to be compared, an initial synchronizing time for all channels was necessary. This was achieved by calculating the compound amplitude at each latency. The compound amplitude was defined as the summation of the absolute values of the amplitudes of each recording channel at the latency in question. Further synchronization was then implemented for each recording channel in order to quantify the similarity between waveforms. Hence, for any two recordings to be compared, synchronization was carried out in a two stage process (Fig. 1):
      • 1)
        The two recordings were shifted and synchronized at the time of the largest compound amplitudes (“initial synchronizing time”) (Fig. 1A). After that, SPES artifact was removed by flattening the record 150 ms before and after the peak of the stimulus artifact. The averaged waveforms were smoothed with a moving average of 20 ms (1/50 of the sample rate) (Fig. 1B).
      • The initial synchronizing time provides a time baseline that takes into account all channels from which the following step will start.
      • 2)
        A second optimized synchronization was calculated for each channel with the data window between 500 ms before and 1500 ms after the initial synchronizing time (Fig. 1C) in order to quantify the similarity between waveforms of homonymous channels. For homonymous recording channels, the correlation coefficients and their significance values were calculated after successive one-sample time shifts of one of the two recordings to be compared. This was carried out for time shifts between 175 ms before and after the initial synchronizing time. For each channel, the time shift used to yield the highest correlation coefficient was the “final synchronizing time.” Values of the final synchronizing time between 70 ms before and 100 ms after the initial synchronizing time included all values between 10th and 90th percentiles of all final synchronizing times. Homonymous channels with a final synchronizing time beyond these limits were considered “dissimilar” channels.
      Figure thumbnail gr1
      Figure 1Four samples of the signal processing steps. For each step, the correlation coefficient (r) and the least square difference (LS) are shown. First, for each channel, an averaged K-complex and an averaged SPES response are shifted and synchronized at initial synchronizing time (A). After that, SPES artifact is removed (B). The second optimized synchronization (final synchronizing time) is calculated for each channel (C) and amplitudes are adjusted (D). Different situations are reflected in each sample. Sample 1 represents an example of high similarity between the K-complex and the SPES response. Sample 2 also shows similar waveforms after some time shifting. Sample 3 reflects different waveforms that share a restricted similarity due to a positive deflection (downwards) in a segment of the recording. Sample 4 illustrates the case of very different waveforms that even reaches the limit of time shifting (step c).
      In summary, the final synchronizing time is defined for each channel, and is an indication of the degree of time shifting from the overall (initial) synchronizing time which is required to obtain the highest correlation for each channel.
      As the correlation coefficient does not take into account signal amplitude, traces markedly different in amplitude can misleadingly yield a high correlation coefficient. To minimize this effect, once each channel was synchronized at the final synchronizing time, amplitudes were adjusted to yield the least square difference between waveforms (Fig. 1D). Amplitude adjustments consisting of increments by a factor of 3.3 or decrements by 0.3 (i.e. more than 70% of the largest amplitude) included all values between the 10th and 90th percentiles of all least square differences. Consequently, if amplitude adjustments greater than 70% of the largest were required, channels were considered too different to resemble each other (see below) and therefore considered “dissimilar” channels. This prevented low amplitude background noise from being selected for analysis.
      Homonymous channels were considered “similar” to one another where the following three criteria were met:
      • a)
        The final synchronizing time occurred between 70 ms before or 100 ms after the initial synchronizing time.
      • b)
        The difference in amplitude between both channels was less than 70% of the largest.
      • c)
        The highest correlation coefficients exceeded 0.5 with P < 0.01.
      In order to quantify the similarity between two recordings, we have defined the similarity index (SI) between two recordings as the percentage of similar homonymous channels. SPES responses were deemed similar to K-complexes (K-complex like responses) if the overall SI exceeded 50%, i.e. 50% or more of homonymous channels were similar.
      In a final phase of analysis, stimulation sites were grouped into regions: orbitofrontal, lateral frontal, parietal, temporal, rostral anterior cingulate and dorso-caudal anterior cingulate. The proportion of similar SPES responses and K-complexes elicited by stimulation of each region was calculated. The differences between these proportions were analyzed using a chi-square test.

      Results

      Patients

      Out of the initial 269 patients, the inclusion and exclusion criteria identified 6 patients (2 men, 4 women; median age 35; range 24–49). Their characteristics are shown in Table 1. Patients had between 6 and 10 electrode bundles, ranging from 45 to 68 electrodes per patient. Figure 2 shows all electrodes implanted in all 6 patients. All patients had electrodes located in medial and lateral frontal areas bilaterally. In addition, 4 patients had additional electrodes located in orbitofrontal areas, 3 had electrodes located in temporal areas and 1 in the parietal lobe. Intracranial recordings in all 6 patients were obtained with reference to a midpoint between Cz and Pz.
      Table 1Patients details. Seizure onset was established according to the intracranial recordings.
      PatientsSexAgeSeizure onsetResectionPathology after resectionNumber of channelsHighest SIStim
      1F34L Lateral frontal cortexL FDNET6072%L AC
      2F39UndeterminedL MFFCD type 24295%L AC
      3M32L Lateral frontal cortexNoneNA5375%L AC
      4M49L Medial frontal cortexL FFCD type 24958%L AC
      5F36UndeterminedNoneNA6278%R AC
      6F24R Medial frontal cortexR MFFCD type 26078%R AC
      L = Left, R = Right, F = Frontal, MF = Medial Frontal, NA = Not Applicable, DNET = Dysembryoplastic neuroepithelial tumor, FCD = Focal cortical dysplasia, SI = Similarity index, Stim = Site stimulated to induce the response with highest SI when compared with averaged K-complexes, AC = Anterior cingulate gyrus.
      Figure thumbnail gr2
      Figure 2Spatial distribution of implanted sites (white circles) in all patients. R = Right, L = Left.
      During sleep, all six patients showed K-complexes.
      Four patients subsequently underwent frontal resections for the treatment of epilepsy. Histopathology revealed Taylor type focal cortical dysplasia (type 2) in 3 subjects and a dysembryoplastic neuroepithelial tumor in one.

      Similarity between K-complexes and SPES responses

      SPES responses occurred after each stimulus at 98.5% of stimulated sites and were highly stereotyped across repeats of identical stimulation at each recording electrode. Conversely, the morphology, amplitude and distribution of SPES responses varied markedly with the stimulated region, ranging from low amplitude deflections to widespread, bilateral responses. Examples are shown in Figure 3, Figure 4.
      Figure thumbnail gr3
      Figure 3Example of response similar to K-complex from patient 2 after stimulation of dorso-caudal anterior cingulate. Top: Resemblance between averaged spontaneous K-complex (left) and SPES response (right). The column between the two recordings shows the correlation coefficient (r) between homonymous channels, showing values of up to 0.97. Bottom: The red dot shows the stimulation site used to induce the SPES response with the highest SI when compared to K-complexes and the yellow dots show all EEG recording sites. The pie chart shows the percentage of homonymous channels with correlation coefficients between 0 and 0.25 (green), 0.25 and 0.50 (yellow), 0.50 and 0.75 (orange), 0.75 and 1 (red). The percentage of dissimilar channels (see section) appears in blue. R = Right, L = Left, r = Correlation coefficient. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
      Figure thumbnail gr4
      Figure 4Example of response not similar to K-complex from patient 2 after stimulation of rostral anterior cingulate. Top: Resemblance between averaged spontaneous K-complex (left) and SPES response (right). The column between the two recordings shows the correlation coefficient (r) between homonymous channels. Bottom: The red dot shows the stimulation site used to induce the SPES response with the highest SI when compared to K-complexes and the yellow dots show all EEG recording sites. The pie chart shows the percentage of homonymous channels with correlation coefficients between 0 and 0.25 (green), 0.25 and 0.50 (yellow), 0.50 and 0.75 (orange), 0.75 and 1 (red). The percentage of dissimilar channels (see section) appears in blue. Note that most channels were considered dissimilar due to the amplitude or final synchronizing time criteria detailed in methods, despite showing r values of up to 0.61. R = Right, L = Left, r = Correlation coefficient. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

      Visual analysis

      Stimulation at sites in the anterior cingulate frequently resembled sleep K complexes. Conversely, stimulation of lateral frontal cortex, posterior cingulate, orbitofrontal cortex, temporal lobe (including hippocampal) or parietal regions did not readily elicit responses resembling K-complexes.

      Quantitative analysis

      Figure 5 shows the stimulation sites that induced SPES responses with SI (similarity index, see Methods) exceeding 50% (n = 14) when compared to sleep K-complexes. All responses showing SI above 50% were induced by stimulation of the frontal cortex, and most were in the anterior cingulate gyrus. By contrast, no responses induced by stimulation of the posterior cingulate, temporal or parietal regions showed responses with SI above 50%.
      Figure thumbnail gr5
      Figure 5Sites whose stimulation induced SPES responses with SI above 50% (mean = 69.49%, SD = 10.37) when compared to averaged K-complexes in all patients. Red dots represent stimulation sites which elicit the highest SI between SPES responses and K-complexes in each patient (mean = 75.80%, SD = 12.22). R = Right, L = Left. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
      Among the 14 recordings with SI above 50%, the median SI was 69.49% (SD = 10.37). In some cases, similarity to K-complexes was as high as 81% of channels exhibiting correlation coefficients >0.75 (Fig. 3). Moreover, for each patient, the stimulation site which elicited the highest SI between SPES responses and K-complexes was located in the anterior cingulate gyrus or in the underlying white matter (Figure 5, Figure 6); more specifically, in or by the dorso-caudal half of the anterior cingulate gyrus (red dots Fig. 5, SI: mean = 75.80%, SD = 12.22).
      Figure thumbnail gr6
      Figure 6Averaged intracranial K-complexes from each 6 patients displayed in referential montages and topography of the electrodes used to induce the most similar K-complex-like responses. Each graph shows recordings from the electrode bundle used to stimulate the site that induced the responses resembling K-complexes the most. The electrodes used to induce such responses are shown in red. For each electrode bundle, electrode 1 is the deepest, which is located at the anterior cingulate. In patient 1 electrode 4 was defective and therefore the corresponding channel has been removed from the figure. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
      The median amplitude of K-complexes was 464.57 μV (range 203.56–772.41 μV) and that of the most similar SPES responses was 642.68 μV (range 192.07–793.77 μV).
      All patients remained awake during stimulation of the regions inducing K-complex-like responses.
      Table 2 summarizes the degree of similarity between K complexes and SPES responses when stimulating at different regions. For each stimulated region, Table 2 shows the number and proportion of homonymous channels meeting the criteria for similarity (see Methods section). Responses to stimulation of the dorso-caudal anterior cingulate (n = 24) showed the highest proportion of similar channels (63.33%). All other regions bore less than 35% of channels exhibiting similarity. Chi-square analysis revealed the existence of differences in the proportion of similar channels among regions (P < 0.0001; 63.33% observed vs 35.88% expected), which attributable to the increased proportion of similar channels when stimulating at the dorso-caudal anterior cingulate.
      Table 2Stimulation sites and similarity between homonymous channels.
      Stimulated regionNo. of stimulation sitesNon-similar recording channelsSimilar recording channels
      Orbitofrontal8372 (80.69%)89 (19.31%)
      Rostral anterior cingulate291094 (74.78%)369 (25.22%)
      Dorso-caudal anterior cingulate24454 (36.67%)784 (63.33%)
      Lateral frontal4115 (100%)0 (0%)
      Parietal2291 (81.51%)66 (18.49%)
      Temporal6162 (65.85%)84 (34.15%)
      Total73

      Similarity between spontaneous interictal discharges and SPES responses

      Two patients (patients 2 and 6 in Table 2) exhibited interictal epileptiform discharges during wakefulness that resembled each patient's K-complex (SI: 55.54% and 70.00% respectively). Of note, both were subsequently found to have histology-confirmed focal cortical dysplasia in the anterior cingulate gyrus, in contrast to the other four patients.

      Discussion

      We have found that electrical stimulation of the frontal lobes while awake can induce responses similar to sleep K-complexes. The responses of greatest similarity are elicited by stimulation of the anterior cingulate gyrus or its underlying white matter (red dots in Fig. 5). Their similarity is apparent both visually and quantitatively. Stimulation of other regions including the posterior cingulate, orbitofrontal, medial frontal, temporal, hippocampal or parietal areas did not induce such responses. Our findings are consistent with a model in which the dorso-caudal anterior cingulate initiates, and possibly coordinates, the widespread synchronous cortical process that constitutes the K-complex. Indeed, slow waves and K-complex-like responses can also be evoked by cortical transcranial magnetic stimulation over sensorimotor areas [
      • Massimini M.
      • Ferrarelli F.
      • Esser S.K.
      • et al.
      Triggering sleep slow waves by transcranial magnetic stimulation.
      ]. However, previous studies with intracranial EEG recordings in humans have suggested that the cingulate cortex and functionally related mesial frontal structures appear uninvolved in generating the visible human K-complex waveform [
      • Wennberg R.
      Intracranial cortical localization of the human K-complex.
      ]. This discrepancy is most likely due to differences in methodology: whereas EEG localization identifies the generator of activity at a given time, electrical stimulation used in the present work identifies the structure involved in “initiating” the course of events that will generate the K-complex.
      K-complexes are thought to be related to sleep arousals rather than sleep generation. Therefore it is perhaps not surprising that K-complex-like responses can be induced by unilateral stimulation in awake patients without inducing sleep. This suggests that cingulate stimulation activates only part of the circuits involved in sleep, probably sparing the subcortical loops. This supports that K-complex generation largely relies on cortico-cortical connections. Indeed, the slow cortical oscillations involved in the genesis of K-complexes [
      • Amzica F.
      • Steriade M.
      The functional significance of K-complexes.
      ] appear to be intrinsically generated in cortical neurons [
      • Steriade M.
      • Nuñez A.
      • Amzica F.
      A novel slow (<1 Hz) oscillation of neocortical neurons in vivo: depolarizing and hyperpolarizing components.
      ]. Moreover, there are profuse bilateral connections between medial frontal cortices [
      • Lacruz M.E.
      • García Seoane J.J.
      • Valentin A.
      • Selway R.
      • Alarcón G.
      Frontal and temporal functional connections of the living human brain.
      ], which may be responsible for the bilateral cortical involvement seen during both, normal sleep K-complexes and electrically induced K-complexes [
      • Amzica F.
      • Steriade M.
      Short- and long-range neuronal synchronization of the slow (<1 Hz) cortical oscillation.
      ]. In the generation of normal sleep K-complexes, subcortical structures could modulate cortical excitability, allowing for neuronal synchronization similar to that induced by focal stimulation of the anterior cingulate in our awake patients.
      The neurochemical and electrophysiological state of the brain is very different between sleep and awake states. The fact that we can induce “sleep” phenomena while awake suggests that some sleep mechanisms are somehow present but “latent” during wakefulness, and can be activated by stimulation of the appropriate site while awake. Moreover, it is possible that by stimulating the cingulate gyrus we are activating only a sub-branch of the “K-complex” pathway. In that case we would expect only a channel subset within the SPES response to simulate K-complexes in contrast to what we have observed.
      Other relatively large-amplitude evoked slow responses arising from medial cortical structures (such as the P300) during wakefulness have been proposed as a marker of conscious perception [
      • Sergent C.
      • Baillet S.
      • Dehaene S.
      Timing of the brain events underlying access to consciousness during the attentional blink.
      ,
      • Dehaene S.
      • Changeux J.P.
      Experimental and theoretical approaches to conscious processing.
      ], a proposal that has been recently challenged by evidence suggesting that these responses are absent when conscious perception is present [
      • Melloni L.
      • Schwiedrzik C.M.
      • Müller N.
      • Rodriguez E.
      • Singer W.
      Expectations change the signatures and timing of electrophysiological correlates of perceptual awareness.
      ,
      • Pitts M.A.
      • Metzler S.
      • Hillyard S.A.
      Isolating neural correlates of conscious perception from neural correlates of reporting one's perception.
      ]. Our results, showing that large slow responses can be elicited by SPES of the frontal cortex without subjects being conscious of the stimulus, provide new insight in this debate as they may represent a more general default-mode of reactivity.
      Anterior cingulate stimulation consistently induced K-complex like responses but not sleep spindles. This suggests that the physiology of K-complexes is independent from that of sleep spindles. In contrast to K-complexes, spindles are expressed in the cortex via thalamo-cortical excitatory projections [
      • Contreras D.
      • Esteriade M.
      Cellular basis of EEG slow rhythms: corticothalamic relationships.
      ] and K-complexes appear to trigger sleep spindles probably due to connections between cortex and the reticular nucleus of the thalamus. However, our findings suggest that during wakefulness, cingulate stimulation is unable to activate these loops in a fashion which induces sleep or spindles.
      This study with intracranial electrodes was necessarily limited to patients with epilepsy. Anti-epileptic medication has been shown to reduce the abundance of K-complexes [
      • Colrain I.M.
      The K-complex: a 7-decade history.
      ,
      • Johnson L.C.
      • Hanson K.
      • Bickford R.G.
      Effect of flurazepam on sleep spindles and K-complexes.
      ,
      • Legros B.
      • Bazil C.W.
      Effects of antiepileptic drugs on sleep architecture: a pilot study.
      ] but to date there is no evidence that their morphology or distribution is altered [
      • Wennberg R.
      Intracranial cortical localization of the human K-complex.
      ]. The question of whether epilepsy itself may have influenced our findings is more complex. In our series, seizure onset in 4 out of 6 patients did not involve medial frontal cortex, suggesting that our observations are not necessarily due to abnormalities in cingulate cortex. Responses to SPES and spontaneous epileptiform discharges show similar characteristics in terms of cellular behavior [
      • Alarcón G.
      • Martinez J.
      • Kerai S.V.
      • et al.
      In vivo neuronal firing patterns during human epileptiform discharges replicated by electrical stimulation.
      ], EEG morphology [
      • Nayak D.
      • Valentín A.
      • Selway R.P.
      • Alarcón G.
      Can single pulse electrical stimulation provoke responses similar to spontaneous interictal epileptiform discharges?.
      ,
      • Valentin A.
      • Lazaro M.
      • Mullatti N.
      • et al.
      Cingulate epileptogenesis in hypothalamic hamartoma.
      ] and cognitive effects [
      • Lacruz M.E.
      • Valentín A.
      • Seoane J.J.G.
      • Morris R.G.
      • Selway R.P.
      • Alarcón G.
      Single pulse electrical stimulation of the hippocampus is sufficient to impair human episodic memory.
      ]. Both appear to share similar generic physiological mechanisms and spontaneous interictal epileptiform discharges could be considered as triggered by some form of endogenous stimulation or synchronization. Therefore, it is possible that lesions at the region whose stimulation induces K-complexes can also originate interictal discharges similar to K-complexes, as observed in two of our patients with anterior cingulate cortical dysplasia.
      The beauty of this study is that the localizing power is not only provided by the EEG itself, but also by the stimulation site, i.e. the initiator of a spontaneous event is identified by the site whose stimulation induces a response similar to the spontaneous event in question, making localization independent from the montage used.
      It could be argued that our patients had a predominance of frontal electrodes compared to other lobes (Fig. 2). Patients with bilateral frontal electrodes were deliberately chosen because stimulating at extra-frontal structures in the total population of 269 patients did not induce responses remotely similar to K-complexes. For instance, SPES responses to hippocampal stimulation induces bilateral responses only in 5% of patients [
      • Lacruz M.E.
      • García Seoane J.J.
      • Valentin A.
      • Selway R.
      • Alarcón G.
      Frontal and temporal functional connections of the living human brain.
      ,
      • Jiménez-Jiménez D.
      • Abete-Rivas M.
      • Martín-López D.
      • et al.
      Incidence of functional bi-temporal connections in the human brain in vivo and their relevance to epilepsy surgery.
      ], and when they occur, they are grossly asymmetrical. Furthermore, even within the frontal lobes, the similarity between SPES responses and K-complexes is highly specific of stimulation of the dorso-caudal anterior cingulate or its underlying white matter (Table 2). In any case, the cortical region within the anterior cingulate responsible for K-complex like responses appears to be rather specific. For instance, in patients 1 and 6, stimulation of the deepest contacts of the bundle located at the anterior cingulate did not induce the responses with highest similarity to K-complexes, despite being located in regions apparently similar to those of the remaining four patients (Fig. 6). However, in these two patients, SPES responses most similar to K-complexes were induced when stimulating the white matter underlying the anterior cingulate, which could be explained by stimulation of axons projecting to the anterior cingulate gyrus.

      Conclusions

      This study has implications for sleep physiology. Our findings provide the first causal evidence that the anterior cingulate gyrus initiates widespread synchronous activity that resembles K-complexes. Moreover, cingulate stimulation can induce responses similar to K-complexes during wakefulness, suggesting that subcortical structures may not be required for initiating K-complexes.

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