MICHAEL-FORUM

The MICHAEL-FORUM

Purpose and scope of the Michael Forum

Since 1975 the MICHAEL FOUNDATION has awarded the biennial MICHAEL PRIZE for Epilepsy Research to international clinical and basic scientists.
The MICHAEL FORUM was initiated in order to enable the MICHAEL PRIZE awardees to discuss their research in an informal setting organized every two years in the proximity of larger European scientific conferences. In these workshops, awardees, trustees of the MICHAEL FOUNDATION, the international jury, as well as representatives of the sponsors meet for two days to have an in-depth and at times controversial discussion of the newest findings illuminating causes, consequences, and the treatment of epileptic seizures.

MICHAEL-FORUM 2012

From October 4th to 6tth, 2012, Berlin hosted the 11th edition of the MICHAEL FORUM. The special occasion of the 50th anniversary jubilee of the MICHAEL FOUNDATION brought together 24 Michael laureates to discuss the latest advancement in epilepsy research.

A broad range of exciting topics was covered which can be subdivided into several research areas:

Eleonora Aronica, Dep. of (Neuro) Pathology, University of Amsterdam, The Netherlands
Inflammation in human focal epilepsy: role of astrocytes in the regulation of the immune response

Astrocytes, the major glial cell type of the central nervous system (CNS), are known to play a major role in the regulation of the immune/inflammatory response in several human CNS diseases, including epilepsy. In epileptic human brain tissue, astrocytes undergo significant changes in their physiological properties including the activation of inflammatory pathways. Accumulating experimental evidence indicates that proinflammatory molecules can alter glio-neuronal communication and can contribute to the generation of seizures and seizure-related neuronal damage. In this context, understanding of the astroglial inflammatory response in the epileptic brain and the mechanisms underlying its regulation may provide new strategies to target astrocyte-mediated epileptogenesis

Massimo Avoli, Neurological Institute McGill University, Montréal, Canada
Relationships between interictal- and ictal-like discharges in limbic areas
of in vitro brain slices during 4AP

Rodent limbic areas in the in vitro brain slice preparation can generate interictal- and ictal-like discharges. Here we characterized the epileptiform activity induced by 4-aminopyridine (4AP, 50 µM) by using field potential recordings in the rat entorhinal cortex maintained in vitro and found that: (i) interictal events have a wide range of duration (0.4 - 3.3 s) and interval of occurrence (1.4 - 84.0 s); (ii) ictal discharges can be either shortly preceded by an isolated “slow” interictal discharge (ISID; duration= 1.5 ± 0.1 s, interval of occurrence= 33.8 ± 1.8 s) or initiate suddenly from a pattern of frequently occurring polispike interictal discharge (FPID; duration= 0.8 ± 0.1 s; interval of occurrence= 2.7 ± 0.2 s); (iii) ISID-triggered ictal-like events have longer duration (121.9 ± 5.7 s) and interval of occurrence (383.0 ± 34.8 s) than those initiating suddenly during FIPD (58.3 ± 7.8 s and 202.1 ± 21.8 s, respectively); (iv) high-frequency oscillations (HFOs, 80-500 Hz) are more frequently recorded during ISIDs as compared to FPIDs; and (v) are mostly seen at the onset of ISID-triggered ictal discharges. We conclude that the occurrence of ISIDs along with HFOs during the interictal period may define ictal onset characteristics that are remiscent of those seen in vivo when seizure onset is characterized by low-voltage fast activity. Remarkably, ictal discharges presenting with these features are more robust than those arising suddenly during FPIDs. A similar condition may occur in vivo both in animal models and in humans.

Christophe Bernard, Université de la Mediterranée, Marseille, France
On the Nature of Seizures

Any “normal” brain can be provoked into seizures, e.g. following a convulsive electroshock in human, or following the injection of various chemical compounds in experimental models of epilepsy. Seizures thus belong to the dynamic repertoire of possible brain activities. Their relative stereotypy across species, from human to fruit flies, and across different brain regions, suggests the existence of generic laws governing their dynamics, because they are conserved across very different neuro-architectures. Generic laws have proven very successful in physics through the formulation of canonical models of diverse phenomena including ferro magnetism, lasers and superconductivity. It is in this sense that we wish to understand the nature of seizures by uncovering their canonical features. We demonstrate that a seizure can be fully described by 5 state variables, and unravel the topology of SLEs in their parameter space. This phenomenological approach led to predictions, regarding seizure onset, time course and offset, which were verified experimentally.

Alon Friedman, Ben-Gurion University of the Negev, Beer-Shevam , Israel
Blood-brain barrier functions, network excitability and drug resistant epilepsy

The Blood-brain barrier (BBB) is a complex structural and functional barrier controlling and maintaining a distinct extracellular environment within the brain. In the last decade experimental data has been accumulating revealing the effects of BBB dysfunction on brain functions. Here I will summarize physiological experiments performed in rodents in-vivo and in-vitro in which the brain was exposed to “serum-like” conditions. Our data indicate that proper function of the BBB is crucial for the maintenance of normal neuronal excitability, synaptic transmission and synaptic plasticity. In addition, the protection of the BBB from serum proteins is a key signaling mechanism in controling glia functions including brain immune response and normal vascular response to neuronal activation. Finally, a proper BBB function is a key to pharmacotherapeutics of both peripheral and centrally-acting drugs: BBB opening may allow the delivery of peripherally acting drugs into the central nervous system, causing unwanted neurological signs and symptoms. Including seizures. BBB dysfunction may also alter the effect of brain-permeable drugs and contribute to pharmacoresistance. These new insights into BBB function and dysfunction highlight the importance of standard imaging protocols for the reliable diagnosis of BBB dysfunction in epilepsy and other neurological diseases.

Uwe Heinemann , Inst Neurophysiology Charité Universitätsmedizin, Berlin, Germany
Effects of sK channel agonists on epileptiform activity in human and chronic epileptic tissue

The aim of the present study was to examine parenchymal drug resistance in the temporal cortex of drug resistant epilepsy patients undergoing surgical treatment of epilepsy and to use that tissue for exploration of potential new anticonvulsant principles We investigated acute neocortical human slices and focused on self-sustaining seizure-like events, readily induced by perfusion with artificial cerebrospinal fluid, containing 8 mM potassium and 50 µM bicuculline (121 out of 140 slices from 47 patients). After administration of one anti epileptic drug (either CBZ VPA or PHT) in a sub-sample of 75 slices, seizure-like events persisted in 82.7 % of slices, were replaced by recurrent epileptiform transients in 10.7 %, or became suppressed in 6.7 %, whereas after administration of drug transport inhibitors verapamil and probenecid seizure-like events continued in 60 % of slices, converted to recurrent epileptiform transients in 36 %, and were suppressed in 4 %. Our results suggest that inhibition of P-glycoprotein and multidrug resistance associated proteins modify drug responses but rarely reverse drug resistance in resected cortical tissue. Having established human tissue as a source for acute pharmacological studies we investigated effects of adenosine and of sK channel agonists. While adenosine had relatively weak effects in pilocarpine treated tissue it was more potent in human tissue. By contrast sK channel agonists were effective in both human and pilocarpine treated tissue suggesting that sK channel activation might be a principle from which human patients might profit.

Gregory L. Holmes, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA
Cognition Rehabilitation Following Early-life Seizures: The Power of Oscillations

Cognitive impairment is an often devastating co-morbidity of early life epilepsy and in some children cognitive impairment may be of greater consequence than the epilepsy itself. There is increasing recognition that cognitive co-morbidity can be both chronic primarily due to the underlying etiology of the epilepsy and evolving because of recurrent seizures, interictal spikes and antiepileptic drugs. A myriad of morphological and physiological changes can occur with repeated seizures and determining which, if any of these changes following seizures relate to life-long cognitive and behavioral deficits and to develop interventional techniques.
While it is known that remedial training can help restore cognitive function following early-life seizures, the neural mechanisms of this recovery in memory systems are largely unknown. To examine this issue we measured electrophysiological oscillatory activity in the hippocampus and prefrontal cortex of adult rats that had experienced repeated seizures in the first weeks of life, while they were remedially trained on a memory task. Seizure-exposed rats showed initial difficulties learning the task but performed similar to control rats when increasing delays were imposed. Examination of dynamic oscillation patterns during performance of the memory task revealed that prefrontal cortex theta power was increased among seizure-exposed rats. This enhancement appeared after the first memory training steps (short delays) and plateaued at the most difficult steps (short and long delays). Further, seizure rats showed enhanced CA1-prefrontal theta coherence in correct trials compared to incorrect trials when long delays were imposed, suggesting increased hippocampal-prefrontal synchrony for the task in this group when memory demand was high. Adapted involvement of hippocampal-prefrontal circuits following early-life seizures may underpin cognitive rehabilitation following neurological insults to higher cognitive systems.

Chrysanthy Ikonomidou, Department of Neurology, University of Wisconsin, Madison WI, USA
The evolving role of matrix metalloproteinases in seizures and epileptogenesis

Matrix metalloproteinases (MMPs) are extracellular matrix proteases involved in tissue repair, cell death and morphogenesis. Evidence has been generated implicating involvement of matrix metalloproteinases in epilepsy and epileptogenesis. Our own work in this area has focused on the involvement of MMPs, especially MMP9, in trauma- and seizure-induced cell injury in infancy. The results suggest a substantial contribution of MMP-9 to cell death after traumatic brain injury and after pilocarpine-induced seizures in the developing brain. I will present an overview on the current state of knowledge about involvement of MMPs in seizure-induced cell death and epileptogenesis.

Dieter Janz, Berlin, Germany
Long term seizure outcome in juvenile myoclonic epilepsy

Early studies suggested that JME is easy to control with antiepileptic drugs (AED) but cure with complete remission after withdrawal of AED seemed unlikely ever to achieve.
Because oft the high relapse rate after short and medium time treatment most authors concluded that AED treatment should be continued lifelong.
Recently four longitudinal studies were performed, two with periods of 19.6 and 25.8 years (Baykan et al. 2008, Camfield and Camfield 2009) and two with periods of 39.1 and 44.6 years (Geithner et al. 2012, Senf et al. 2012). The last two studies performed in Greifswald and in Berlin with 31 and 66 patients described a remission rate (remission during the terminal 5 years) in 67.7% and 59.1%. The relatively low rate of relapses after AED withdrawal (3 of 6 and 3 of 14) and the corresponding rate of “cured“ (at least 5 years remission after AED withdrawal) patients (altogether 17 of 23) is most likely explained by the long observation period.
Thus, for the long term prognosis of JME there seems to be a good chance to stay seizure free without AED treatment.

Matthias Koepp, Nat. Hospital for Neurology and Neurosurgery, London, United Kongdom
Evidence to support the hypothesis of multidrug transporter overexpression
as a mechanism of pharmaco-resistance

We present studies with [11C]verapamil PET in healthy controls showing that tariquidar-induced Pgp modulation at the human (BBB) appeared to be transient and its magnitude directly proportionate to serum drug exposure. In patients with temporal lobe epilepsy (TLE), the K1 (influx rate constant) values from drug-sensitive patients were higher than those from drug-resistant patients falling, which suggests more efficient P-gp function in drug-refractory patients resulting in lower drug concentrations in the brain. This difference was restricted to the temporal lobes. After TQD administration, we observed smaller K1 increases in the drug-resistant patients compared to healthy volunteers supporting over expression of Pgp function, with the ipsilateral epileptogenic hippocampus being most significantly affected. VPM uptake after Pgp inhibition with tariquidar in the hippocampus correlated with Pgp-immunopositive labeling in pharmaco-resistant TLE patients who underwent anterior temporal lobe resection for surgical treatment. Our findings provide evidence supporting the hypothesis of multidrug transporter overexpression as one important mechanism for developing pharmacoresistance. The availability of imaging biomarkers will support the development of new treatment strategies targeted at multidrug transporter and aimed at reversing pharmacoresistance with selection of optimal patients and assessment of molecular targets. This will lead to improved health care through individualised treatment strategies, and at the same time to a reduction of health care costs by discontinuing ineffective therapies.

Rüdiger Köhling, University of Rostock, Germany
After-hyperpolarizing potentials (AHP) in the control of neuronal epileptic excitability

In hippocampal CA1 neurons, a prolonged depolarization evokes a train of action potentials followed by a prominent afterhyperpolarizing potential (AHP) which critically dampens neuronal excitability. Since it is not known whether epileptiform activity alters the AHP, and whether any alteration of the AHP is independent of inhibition, we acutely induced epileptiform activity by bath application of the GABAA receptor blocker gabazine (5 μM) and studied its impact on the AHP using intracellular recordings. Following 10 minutes of gabazine wash-in, slices started to develop spontaneous epileptiform discharges. This disinhibition was accompanied by a significant shift of the resting membrane potential of CA1 neurons to more depolarized values. Prolonged depolarizations (600 ms) elicited a train of action potentials, the number of which was not different between baseline and gabazine treatment. However, the AHP following the train of action potentials was significantly reduced after 20 minutes of gabazine treatment. When the induction of epileptiform activity was prevented by co-application of 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX, 10 μM) and D-(- )-2-amino-5- -amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) and N methyl-D-aspartate (NMDA) receptors, respectively, the AHP was preserved despite of GABAA receptor inhibition suggesting that the epileptiform activity was required to suppress the AHP. Moreover, the AHP was also preserved when the slices were treated with the protein kinase blockers H-9 (100 μM) and H-89 (1 μM). These results demonstrate that the AHP following a train of action potentials is rapidly suppressed by acutely induced epileptiform activity due to a phosphorylation process - presumably involving protein kinase A.

G K Kostopoulos, Medical School, University of Patras, Greece
Understanding sleep mechanisms contributes to a redefinition
of the term "generalized“ of absence seizures

Historically the dichotomous classification of "focal" and "generalized" seizures and their epilepsies has been as much clinically demanded as neurophysiologically debated. Lately the term generalized is no longer an obligatory characterization of epilepsies, while for their seizures the idea of a generalization from the start has been gradually abandoned in favor of an origin within a local network and rapidly engaging bilaterally distributed cortical and subcortical ones. Considerable support to the new ideas has been given by recent advances on brain mechanisms of sleep. Experiments with animal models of absence seizures with generalized spike and wave discharges (GSWD) had early on demonstrated the importance of cortical hyperexcitability implicating thalamocortical circuits like the ones underlying sleep spindles. Cortical excitability has been recently shown to follow bistable dynamics at all time scales from the circadian sleep/wake, to the sleep cycles and stages, to the cyclic alternating pattern (CAP) of NREM sleep and the slow waves of CAP A-phase, which reflect "down" and "up" states of excitability of single cortical neurons. We have contributed MEG tomographic evidence for localized (mainly medial frontal) activations (Ioannides et al., 2009) and for a very dynamic relationship between K-complexes and spindles (Kokkinos and Kostopoulos, 2011) during NREM sleep. In patients with JME and CAE, GSWD appeared mainly in CAP-A (considered a result of cortical activation) and the transitions from CAP-A to CAP-B and had different EEG topographic distribution compared to focal interictal and pre-generalization SWD which occurred mainly in CAP-B periods (Koutroumanidis et al 2012). The latter may reflect a system of multifocal non-localizing electrically unstable cortical areas that under the facilitatory influence of exogenous or endogenous factors like sleep instability can foster a corticothalamic response strong enough to generate autonomous 3Hz GSWD.

Wolfgang Löscher, K. Töllner, M. Töpfer, G. Brunhofer, T. Erker, P. W. Feit and C. Brandt,
University of Veterinary Medicine Hannover and University of Vienna, Germany / Austria
Anticonvulsant and antiepileptogenic effects of bumetanide and novel bumetanide derivatives

A variety of brain insults have the potential to induce the development of epilepsy. The development of neuronal hyperexcitability is thought to be a critical event in this process. Cation-chloride co-transporters (CCCs) play an important role in the formation of abnormal excitatory GABAergic neurons and provide a potential target for antiepileptogenic treatment. Different pro-epileptogenic brain insults have been shown to downregulate the K+-Cl- co-transporter KCC2 and upregulate the Na+-K+-2Cl- co-transporter NKCC1 leading to an increase in intracellular Cl- and thus a shift from inhibitory to excitatory GABA actions that may contribute to the development of neuronal hyperexcitability.
The diuretic drug bumetanide is a selective inhibitor of NKCC1 giving the opportunity to counteract the upregulation of NKCC1 during epileptogenesis. We have recently reported that bumetanide exerts disease-modifying effects when administered after a pilocarpine-induced status epilepticus in rats, but these effects only occurred when bumetanide was co-administered with phenobarbital (Brandt et al., J. Neurosci. 30, 8602-8612, 2010). A likely explanation for this observation is that we found that bumetanide, due to its high ionization at physiological pH, does not effectively cross the blood brain barrier (BBB). Furthermore, the half-life of bumetanide in the rat is only about 10 min, so that effective levels are difficult to achieve and maintain in vivo (Brandt et al., 2010).
We therefore started to develop bumetanide derivatives with enhanced brain penetration and prolonged maintenance in the brain. One strategy is to mask the acid group of bumetanide by lipophilic esters, which should penetrate the BBB more easily than the parent drug and act as prodrugs for bumetanide. The feasibility of this strategy was demonstrated by studying the brain uptake of various prodrugs in mice and rats. Subsequent experiments showed that the most promising bumetanide prodrug (BUM5) exerted anticonvulsant and disease-modifying effects in mouse and rat models of epilepsy but was less diuretic than bumetanide, demonstrating the advantages of our strategy.

Solomon L. Moshé, Albert Einstein College of Medicine, New York, USA
Multiple hit model of symptomatic (structural/metabolic) infantile spasms

Dr Solomon L. Moshé discussed the multiple hit model of symptomatic (structural/metabolic) infantile spasms (patent 7863499, Albert Einstein College of Medicine) that has provided us with new insights towards achieving the goal of curing infantile spasms. Following the right intracerebral administration of doxorubicin and lipopolysaccharide at postnatal day 3 (PN3), rat pups have spasms between PN4-PN13, followed by the emergence of other seizures after PN9, impaired sociability and increased grooming (stereotypies), as well as neurodevelopmental, learning and memory deficits following the resolution of spasms. Systemic administration of p-chlorophenylalanine (PCPA) at PN5 augments the frequency of spasms. Recent data, collected by Dr Aristea S. Galanopoulou (at Albert Einstein College of Medicine) suggest that this model is well qualified for the preclinical screening of new therapies for IS with disease-modifying potential, given that: its phenotype is evolving and amenable to testing disease-modification; it is the only chronic model with a known pharmacosensitivity profile towards the recommended treatments for infantile spasms in human babies; and has reliable endpoints that can be used to evaluate the efficacy of tested drugs against spasms, other seizures (antiepileptogenic effects) but also the associated cognitive and neurodevelopmental comorbidities (disease-modifying effects.

Jeffrey Noebels , Departments of Neurology, Neuroscience, Baylor College of Medicine, Houston, USA
Ion Channel Mutations in Heart and Brain: A Mechanism for Sudden Unexpected Death in Epilepsy

Critical progress in the discovery of genes linked to human epilepsies has been made in the last decade, leading to important insights into mechanisms of seizure-related comorbidity. Chief among these is sudden unexpected death (SUDEP), the major cause of mortality in individuals with epilepsy. Cardiac arrhythmia and respiratory deficiency have long been suspected to contribute to sudden unexpected death following seizures. Fresh evidence from the laboratory reveals that KCNQ1, a gene that encodes a ‘cardiac’ potassium channel responsible for a lethal cardiac arrhythmia, long QT interval syndrome (LQTS), is also expressed in brain neurons. Human mutations in this channel produce a combined phenotype of epilepsy, cardiac LQT syndrome, and sudden death, demonstrating the first monogenic mechanism for sudden death in epilepsy (SUDEP). LQT genes are the first in an expanding list of genes altering cardiorespiratory control centers that may impact SUDEP risk. The NIH-funded ‘SUDEP Research Consortium’ is a new multisite research program planning to integrate a world-wide collection of SUDEP and near-SUDEP cases and their families with clinical and scientific research in order to discover clinically useful biomarkers and therapies to prolong the lives of those with epilepsy.

Heidrun Potschka, University of Munich, Germany
Mechanisms of AED transporter regulation in epilepsy

Overexpression of blood-brain barrier efflux transporters, putatively contributing to drug resistance, has been repeatedly described in rodent epilepsy models and in the human epileptic brain. An endothelial NMDA receptor/cyclooxygenase-2/EP1 receptor pathway that induces enhanced functional expression of Pgp was identified as the cause of seizure-associated induction of this efflux transporter in rodent epilepsy models. The same signaling pathway seems to be active in human capillaries prepared from tissue dissected during epilepsy surgery. The data might render a translational basis for targeting approaches to control Pgp.

Ivan Soltez, UC Irvine, USA
Closed-loop system for detecting and using optogenetic methods to respond
to spontaneous electrographic and behavioral seizures in mice

The inherently unpredictable nature of seizures in epilepsy poses an obvious challenge to the development of ideal therapeutic strategies that would act only on an ‘as-needed’ basis without disrupting normal interictal behaviors. It has recently become possible to control specific subsets of neurons utilizing light-sensitive channels or pumps (opsins), allowing for unprecedented specific and immediate control of cell populations of interest in the behaving animal. However, the widespread use of these novel optogenetic technologies has so far been difficult to apply to epilepsy, in part because of the unpredictable nature of the seizures. Here, I will describe the development and use of a closed-loop system for detecting and using optogenetic methods to respond to spontaneous electrographic and behavioral seizures in mice. Our results suggest that diverse on-demand optogenetic strategies can be effective at controlling seizure activity.

E.-J. Speckmann, Münster, Germany
Human amygdala in vitro: New news

The lateral nucleus of the amygdala (LA), major input station of the amygdala, gives rise to most of its projections from the amygdala to the hippocampal area. To date, knowledge on functional mesiotemporal amygdala connections is exclusively derived from animal experiments. Therefore, the present study was aimed to investigate anatomo-functional networks in the human LA by focusing on activity spreading patterns.