Conference and Workshop activities

In an effort to decrease the risk of side effects associated with traditional drug design Work Package 1 (WP1) has been focussed on the mechanism of action of target-specific research drugs and drug candidates. To test action mechanisms, new in silico, in vitro and in vivo methods have been developed. Being pivotal for the development of drugs with reduced side effects and for an increased probability of clinical trial success, new pathomechanism-specific pathways have been disclosed. Based on the knowledge of the three dimensional structure of validated targets in complex with agonists and antagonist, a more comprehensive understanding of target binding and function became available. Disclosing new functions of known or new target subtypes has been provided opportunities for more specific design of drugs and for the development of possible neuroprotective strategies of treatment and therapy.
WP1 performed different types of activity, including laboratory training of PhD students Péter Barabás, Bálint Lasztóczi, László Héja and Tünde Molnár in co-operation with Profs. Arne Schousboe, Hans Bräuner-Osborne (Copenhagen) and Vincenzo Crunelli (Cardiff); research training of Dr. Károly Orbán-Kis (Romania), holding seminars open for PhD students and the scientific community by inviting research expert speakers Dr. Didier Rognan (Strasbourg), Prof. Vincenzo Crunelli (Cardiff), Dr. Tibor Szilágyi (Romania), Prof. Susan Amara (Pittsburgh); organisation of an international workshop on “Free Radicals in Epilepsy” and a nationwide conference with the participation of lecturers from academia and industry on “Design of New Therapeutic Compounds and Diagnostic Methods, Based on Validated Target Molecules”; participation in intramural, national and international workshops, meetings, symposia and conferences by delivering 34 lecture and poster presentations; introduction of new techniques; enhancement of international research co-operations.

Focal epilepsies contribute with up to 70 % to the 30 % of drug resistant epilepsies. The most frequent form of focal epilepsies is mesial temporal lobe epilepsy. Two hypotheses try to ex-plain pharmaco-resistance. The transporter hypothesis suggests that upregulation in MDR´s and MRP´s at the blood brain barrier or in glial cells prevents reaching of sufficient drug con-centrations. The target hypothesis implicates alterations in network, cellular or molecular changes rendering the antiepileptic drugs ineffective. Drug transporter upregulation has been described in a number of temporal lobe epilepsy models and expression of drug transporters has been described in tissue samples from patients with developmental disturbances and in samples from temporal lobe epilepsy tissue where expression was increased in hippocampal tissue from patients with hippocampal sclerosis but less so in tissue from patients with non hippocampal sclerosis. Evidence for the target hypothesis comes from observations on altera-tions in sodium channel properties (the target for phenytoin, carbamazepine and lamotrigene) and from alterations in GABA receptors (the target for GABA mimetic drugs such as barbitu-rates, benzodiazepines, tiagabine and vigabatrine). In addition drug resistance to GABAergic drugs may result from increased interconnectivity between GABAergic cells or from silencing of GABAergic cells due to tonic inhibition or due to upregulation of K channels in such cells.
We have developed models of drug resistant status epilepticus where combined application of 4-AP and bicuculline results in drug resistant status epilepticus or where after prolonged ex-posure to low Mg artificial cerebrospinal fluid seizure like events evolve in drug resistant ac-tivity likely due to increased consumption of GABA in the tricarboxic acid cycle under condi-tions of impaired metabolism.
We further used human specimens obtained from surgical treatment of epilepsies from tumour patients (drug sensitive) and from patients with and without hippocampal sclerosis. We were able to induce epileptiform discharges in such tissue by elevating potassium concentration. The ensuing activity was not blocked by CBZ when the tissue came from patients with drug refractory epilepsy while the epileptiform discharges were blocked in patients, which were not (yet) drug resistant. At present we use blockers of drug transporters to see whether we can reverse insensitivity to CBZ. So far, this was the case only in one of 6 cases. The finding points to the possibility that drug transporters can sometime affect drug resistance but favour alterations in target molecules as the main cause of refractoriness to anticonvulsant drugs .

Somatostatin mediates its diverse physiological effects through a family of four G-protein-coupled receptors (sstr1-sstr4) in the hippocampus. All four receptors have a distinct subcellular distribution. The type II somatostatin receptor sstr1 is primarily localised in axons, thus it functions presynaptically. The sstr2 receptor (type I) is confined to the plasma membrane of neuronal somata, consequently it is postsynaptic. The sstr3 receptor (type I) is selectively targeted to neuronal cilia. Distributed in distal dendrites, sstr4 (type I) is also postsynaptic. The function of individual sstrs is not yet characterised well because of the lack of specific ligands for all subtypes. TT-232, a recently developed 'non-classical' somatostatin analogue containing a five-residue ring structure with a D-amino acid shows a strong in vivo and in vitro antiproliferative effect, induces apoptosis in several tumors and cancer cell lines. In the peripheral sensory nervous system inhibits acute and chronic inflammatory responses and exerts an antinociceptive effect. In this study we characterise its hippocampal effects in vitro.
In CHO cell lines expressing sstr1-sstr4, TT-232 displaced [¹³¹I]Somatostatin in all cell lines with similar affinity. Also, TT-232 activated type 2 extracellular signal-regulated kinase (ERK2) activity in all cell lines. By contrast, effects of somatostatin and TT-232 on low-[Mg²⁺]-induced seizures in the CA3 region of rat hippocampal slices were distinguishable. Somatostatin decreased the length of low-[Mg²⁺]-induced seizures by 18.4±12.9 % and increased the interval between them by 14.1±17.2 %, whereas TT-232 reduced both the length and the interval of low-[Mg²⁺]-induced seizures by 12.9±16.9 % and 27.8±1.2 %, respectively. These results indicated that TT-232 affected only a subset of sstrs. To identify those sstr subtypes that are specifically activated by TT-232, pharmacologically isolated miniature excitatory and inhibitory postsynaptic currents (mEPSC and mIPSC) have been recorded from CA3 hippocampal cells. Somatostatin and TT-232 reduced mEPSC frequency by 32.9±15.9 % and 37.9±10.1 %, respectively, while the sstr2-selective ligand sandostatin had no effect on it. Somatostatin and TT-232 also decreased mEPSC amplitudes by 41.81±65.16 % and 55.2±38.7 %, respectively. Somatostatin reduced the frequency (34.6±18.2 %), but not the amplitude of mIPSCs, while TT-232 and sandostatin did not affect them. In these experiments TT-232 reproduced some of the effects of somatostatin and acted unlike the sstr2-selective ligand sandostatin. This work was supported by grants Center of Excellence on Biomolecular Chemistry QLK2-CT-2002-90436 and NKFP Medichem2 1/A/005/2004.

The GABA neurotransmission system contains a wealth of pharmacological targets for drugs acting on several neurological and psychiatric disorders including epilepsy, pain, anxiety, sleep disorders and possibly schizophrenia. Most of these drugs act on either the receptors, the metabolizing enzyme or the transporters for GABA. One of the drugs in clinical use for treatment of certain types of epilepsy, Tiagabine is a elective inhibitor of the GABA trans-porter subtype GAT1 which is mainly located on GABAergic neurons but is also expressed in astroglial cells. While Tiagabine has only a marginal preference for astroglial GABA transport compared to neuronal transport, a newly developed GABA analog, N-methyl- exo-THPO (3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydro-1,2-benzisoxazol), also selective for GAT1, has a 10-fold preference for inhibition of glial GABA transport compared to neuronal GABA transport and this compound was unexpectedly potent with regard to seizure protection (1). This finding underlines the functional importance of glial GABA transport in GABA homeo-stasis. Recently a large series of lipophilic analogs of exo-THPO (3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazol) has been synthesized and characterized as inhibitors of GABA transport in neurons, astrocytes and cell lines expressing mouse GAT1-4 (2). Among these EF 1502 (N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol), a dithienyl-butenyl derivative of N-exo-THPO, turned out to equipotently inhibit GAT1 and GAT2 (BGT-1). Interestingly it was shown to act synergis-tically with Tiagabine as an anticonvulsant in animal models of epilepsy (3). This points to a hitherto neglected role for the GAT2, betaine transporter in control of seizure activity. As GAT2 is to a large extent located on astroglial cells extrasynaptically, this observation again underlines the importance of glial cells in the maintenance of GABAergic function.

References:
1. White, H.S., Sarup, A., Bolvig, T., Kristensen, A.S., Petersen, G., Nelson, N., Pickering, D.S., Larsson, O.M., Frølund, B., Krogsgaard-Larsen, P. and Schousboe, A. J. Pharmacol. Exp. Therap. 302, 636-644 (2002).
2. Clausen, R.P., Moltzen, E.K., Perregaard, J., Lenz, S.M., Sanchez, C., Falch, E., Frølund, B, Sarup, A., Larsson, O.M, Schousboe, A. and Krogsgaard-Larsen, P. Bioorg. Med. Chem. 13, 895-908 (2005).
3. White, H.S., Watson, W.P., Hansen, S., Slough, S., Sarup, A., Bolvig, T., Petersen, G., Larsson, O.M., Clausen, R.P., Frølund, B., Krogsgaard-Larsen, P. and Schousboe, A. J. Pharmacol. Exp. Therap. 312, 866-874 (2005).

Muscimol is a heterocyclic 3-isoxazolol bioisostere of GABA, showing high affinity for the GABAA receptors. Muscimol is toxic, but it is an interesting lead structure for the design of specific GABAergic drugs. THIP is a specific GABAA agonist, whereas THPO is a specific GABA uptake inhibitor. The isosteric amino acids isonipecotic acid and nipecotic acid were shown to possess potent and highly selective GABAA agonist and GABA uptake inhibitor effects.
THIP is a partial GABAA agonist showing different levels of efficacy at GABAA receptors with different subunit combinations. The zwitterionic structures of muscimol, THIP, and THPO do not prevent these compounds from entering the brain after systemic administration. Zwitterionic THIP enters the brain very quickly, even after oral administration. THIP is well tolerated in man. THIP (under the name Gaboxadol) is now in phase III clinical trials as an agent showing potent non-opioid analgesic effects and an ability to re-establish normal sleep architecture in patients with disturbed sleep patterns.

Amino acids GABA and Glu are the major inhibitory and excitatory signalling molecules in the brain. Clearing GABA and Glu from the extracellular space are known to be mediated distinctly by specific transport proteins (GAT1-GAT4 and EAAT1-EAAT5 subtypes for GABA and Glu, respectively) localised to nerve terminals and surrounding glial cells. Several lines of in vitro evidence provided that a previously unrecognised Glu-sensitive GABA transport mechanism may possibly exist in the brain (Héja et al., J. Med. Chem. 2004, 47, 5620-5629). This mechanism is very much in the forefront of interest by addressing the coexistence of major inhibitory and excitatory neurotransmitters in course of signalling. To explore this mechanism, the non-transportable GABA uptake inhibitor, NNC-711 was used to eliminate the transport activity of the dominant GABA transporter GAT1 present in rat cerebrocortical membrane vesicle fractions and the effect of Glu transport inhibitors on GABA uptake was examined. While the non-transportable Glu uptake inhibitors DL-TBOA and dihydrokainate had no significant effect on the transport process isolated by NNC-711, Glu transporter substrates like L-Glu and t-PDC did inhibit this uptake, even in the presence of 100 µM DL-TBOA. Inhibitors of GABA transporter family members like dopamine and serotonine as well as endogenous amino acids (Leu, Ser) did not have significant effect on this uptake process. The pharmacological profile of GABA transport inhibitors (Β-Ala ~ nipecotic acid >> Tau) suggested that the Glu-sensitive GABA transport process is possibly mediated by a known GABA transporter subtype, either GAT3 or GAT4. In vivo microdialysis data showed contralateral enhancements of both extracellular [Glu] and [GABA] following ipsilateral Β-Ala administration into the rat brain cortex. These findings highlight callosal Glu-sensitive GABA transporter subtypes in shaping interhemispheric inhibition. Growing understanding of the molecular processes underlying the unique Glu-sensitive GABA transport mechanism offers hope for new therapeutic interventions.
Acknowledgement: CBCH QLK2-CT-2002-90436, 1/A/005/2004 NKFP MediChem2 and GVOP-3.1.1.-2004-05-0068/3.0 AKF-050068 Transporter Explorer.

The formation of highly reactive species deriving from molecular oxygen, including free radicals, has been implicated in a large number of pathologies, including ischemia/ reoxygenation injury, neurodegenerative diseases, atherosclerosis or rheumatoid arthritis. In the course of the disease processes, these deleterious species can cause the disruption of the mitochondrial respiratory chains, the release of iron ions from various cellular sites, or some modifications in the activities of key enzymes such as xanthine oxidase. Experimental or clinical studies have focused on the possible contribution of activated oxygen species to tissue injury by measuring the occurrence of membrane peroxidation and destruction, and DNA or proteins oxidation. However, in situations where the initial formation of oxygen-derived free radicals is postulated, analytical methods based on ESR spectroscopy can be decisive. In the present communication, some applications of ESR in in vitro (cerebellar granule cells, isolated perfused organs) or in vivo (myocardial surgery, retinal ischemia reperfusion) models will be presented.

Acknowledgement. This work was supported by the grants from the CNRS (UMR 6517), Ipsen- Beaufour-Pharma, and the S.A.R.L. Oxylab

References:
1. Pietri S, Mercier A, Mathieu C, Caffaratti S, Culcasi M. Hemodynamic and metabolic effects of the beta-phosphorylated nitroxide 2-diethoxyphosphoryl-2,5,5-trimethylpyrrolidinoxyl during myocardial ischemia and reperfusion. Free Radic Biol Med. (2003) 34:1167-77.
2. Muller A, Pietri S, Villain M, Frejaville C, Bonne C, Culcas M. Free radicals in rabbit retina under ocular hyperpressure and functional consequences. Exp Eye Res. (1997) 64:637-43.
3. Lafon-Cazal M, Pietri S, Culcasi M, Bockaert J. NMDA-dependent superoxide production and neurotoxicity. Nature. (1993) 364:535-7

The ESR time scale, where the spectra show dynamic effects is in the order of 105-109 Hz, which range is important for the molecular motions in biological systems. The dynamic information can be extracted by computer simulation of the ESR spectra. Different applications of the method are demonstrated.
1. Superoxy radicals can be trapped by DEPMPO (5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide) yielding to persistent nitroxide radicals. The hindered rotation of OOH group results chemical exchange in the ESR spectra [1].
2. By chemical damage of proteins free radicals are produced and the radicals are observed by spin trapping. To characterise the motion of proteins anisotropic line width tensor is introduced and effective g- and hyperfine tensors are applied. The impact of denaturation is also demonstrated [2, 3].

Acknowledgement. This work was supported by the Center of Excellence on Biomolecular Chemistry (Project CBCH) QLK2-CT-2002-90436.

References:
1. Rockenbauer: Determination of chemical exchange parameters in ESR spectroscopy. Molecular Physics Reports (1999) 26, 117-127.
2. J-L Clément , B.C. Gilbert , A Rockenbauer and P. Tordo. Radical damage to proteins studied by EPR spin-trapping techniques. J. Chem. Soc. Perkins Trans. (2001) 2, 1463-1470.
3. J-L Clément, B.C. Gilbert, A. Rockenbauer, P. Tordo, A.C. Whitwood: Observation of protein-derived (BSA) oxygen-centered radicals by EPR spin-trapping techniques. Free Radical Res. (2002) 36, 883-891

Here we report on a fast kinetics method to monitor the formation of free radicals under epileptic condition. Free radical formation in rat mitochondria was followed by the application of the technique of rapid mixing in combination with fluorescence detection. Aliquots (75 ml) of rat brain mitochondrial suspensions, isolated from sucrose-gradient purified rat cerebrocortical synaptosomes, were rapidly mixed with equal amount of buffer A (containing in millimoles: KCl 140, NaCl 3, CaCl₂ 0.05, EGTA 1, HEPES 10, pH=7.4; control condition) or buffer B (containing, in millimoles: KCl 140, NaCl 30, CaCl₂ 1 , EGTA 1, HEPES 10, pH=7.4; epileptic condition) in the presence of 300 mM dihydroethidium. Dihydroethidium is oxidized by hydroxyl and superoxide radicals to ethidium, showing red fluorescence, monitored at 610 nm using an 530 nm excitation filter [1]. Under these conditions, a two-phased increase in ethidium fluorescence is observed between 0.001-0.01 s and 0.05-0.1 s time intervals, respectively. Fluorescence in both phases decreased in the presence of the radical-scavanger, TEMPO-9AC (0.5 mM).

Acknowledgement. This work was supported by grants 1/ 047 NKFP MediChem, OTKA T 035225, (Hungary), the Center of Excellence on Biomolecular Chemistry (Project CBCH) QLK2-CT- 2002-90436 and Training and Excellence ICA1-CT-2002-70008.

References:
1. Haugland, R. P. 1996. Handbook of fluorescent probes and research chemicals. Leiden: Molecular Probes Inc. pp 483-502.

Increased neuronal activity causes transmembrane ionic shifts. Active and secondary active transport processes are required to restore transmembrane ionic gradients, which depends on delivery of ATP. In neurones most ATP generation depends on oxidative phosphorylation requiring well functioning mitochondria. Previously it was assumed that adaptation of ATP generation to cellular needs is regulated by the ratio of ATP/ ADP. However, some enzymes of the tricarboxilic acid cycle are Ca²⁺ sensitive. We present evidence that Ca²⁺ entry through the plasma membrane leads to mitochondrial Ca²⁺ uptake and increased generation of NADH. We also show that Ca²⁺ release from the endoplasmic reticulum mediated through metabotropic receptors can also trigger NADH generation. Both mechanisms contribute to seizure-associated increased production of NADH which leads to increased ATP production.

Acknowledgement. This work was supported by the grants SFB 507

References:
1. Kann O, Kovacs R, Heinemann U. Metabotropic receptor-mediated Ca2+ signaling elevates mitochondrial Ca2+ and stimulates oxidative metabolism in hippocampal slice cultures. J Neurophysiol (2003) 90:613-21.
2. Heinemann U, Buchheim K, Gabriel S, Kann O, Kovacs R, Schuchmann S.: Coupling of electrical and metabolic activity during epileptiform discharges. Epilepsia. (2002) 43 Suppl 5:168-73.
3. Schuchmann S, Kovacs R, Kann O, Heinemann U, Buchheim K. Monitoring NAD(P)H autofluorescence to assess mitochondrial metabolic functions in rat hippocampal-entorhinal cortex slices. Brain Res Brain Res Protoc. (2001) 7:267-76.

The maintenance of transmembrane ion gradients during epileptic activity represents an extraordinary stress for neurones. Here we addressed the question, whether mitochondria are able to keep up the large ATP demand during seizures. Since the largest part of the proton motive force is represented by the mitochondrial membrane potential (DY), we monitored its changes by fluorescence means at multicellular, single cell and mitochondrial levels during epileptiform activity induced by application of Mg2+-free ACSF in hippocampal slice cultures. Rhodamine-123 (Rh-123) application through the patch pipette in the whole cell mode allowed measurement of changes of DY in mitochondria from the soma and dendrites of a single CA3 pyramidal cell by using confocal laser scanning microscopy. Rh-123 staining showed a complex 7 network of tubular and granular mitochondria with different DY. Upon interictal activity, localised mitochondrial depolarisation occurred in small dendritic branches, often restricted to a single mitochondria. In contrast, seizure-like events (SLEs) were associated with a homogeneous, overall increase of cytosolic Rh-123 fluorescence, indicating simultaneous depolarisation in a large population of mitochondria. However, signs of mitochondrial dysfunction (i.e. sudden, large depolarisation with apparent swelling), known from excitotoxic glutamate application, could be only seen after a sequence of several (>5) recurring SLEs. Mitochondrial depolarisation depended on Ca²⁺ influx, as blockade of the mitochondrial Ca²⁺-uniporter by Ru360 inhibited SLE-associated Rh-123 fluorescence rise. Selective blockade of the electrophoretic mitochondrial Ca²⁺/Na⁺-exchanger by CGP-37157 also inhibited fast Rh-123 fluorescence changes, suggesting that enhanced mitochondrial Ca²⁺-cycling, rather than Ca²⁺-uptake alone, is responsible for the mitochondrial membrane potential changes.

Acknowledgement. This work was supported by grants 1/ 047 NKFP MediChem, OTKA T 035225, (Hungary), the Center of Excellence on Biomolecular Chemistry (Project CBCH) QLK2-CT-2002-90436 and Training and Excellence ICA1-CT-2002-70008 and SFB 507.

References:
1. Kovacs R, Schuchmann S, Gabriel S, Kann O, Kardos J, Heinemann U. Free radicalmediated cell damage after experimental status epilepticus in hippocampal slice cultures. J Neurophysiol. (2002) 88:2909-18.
2. Kovacs R, Schuchmann S, Gabriel S, Kardos J, Heinemann U. Ca2+ signalling and changes of mitochondrial function during low-Mg2+-induced epileptiform activity in organotypic hippocampal slice cultures. Eur J Neurosci. (2001) 13:1311-9.
3. Schuchmann S, Luckermann M, Kulik A, Heinemann U, Ballanyi K. Ca2+- and metabolismrelated changes of mitochondrial potential in voltage-clamped CA1 pyramidal neurons in situ. J Neurophysiol. (2000) 83:1710-21.

ROS (reactive oxygen species)-induced alterations of brain function play an important role under many pathological conditions of the central nervous system including epilepsy. Mitochondrial respiratory chain complexes I and III have been shown to be the main contributors to neuronal ROS production since the superoxide anion, a product of one-electron reduction of oxygen, is the byproduct of normal functioning of mitochondrial respiratory chain. While the superoxide producing site at respiratory chain complex III (b-c1 complex) has been determined to be the bound semiquinone at center ’o’ of Q-cycle the ROS producing site at respiratory chain complex I has not been identified so far. In the present study we approached this question investigating the effects of inhibitors and of redox potential of the NAD system on H2O2 production of isolated rat brain mitochondria applying p-hydrophenylacetate as fluorescent probe. While mitochondria in the presence of glutamate+malate alone do generate only small amounts of hydrogen peroxide a high production is observed after the addition of the complex I inhibitor rotenone or in the presence of the respiratory substrate succinate alone. This is an indication that most of the superoxide radicals are produced at complex I and that a high production of reactive oxygen species is a feature of respiratory chain-inhibited mitochondria or of reversed electron flow. Very importantly, the rate of hydrogen peroxide generation by respiratory chain complex 8 III which can be observed in presence of the complex III inhibitor antimycin A is substantially lower. Furthermore, we determined the redox potential of the superoxide production site at complex I to be -290 mV. This result suggests that the site of superoxide generation at complex I is FMN. Since short term incubation of rat brain mitochondria with H₂O₂ in the lower mM range induced increased ROS production at this site we propose that reactive oxygen species activate a self-accelerating vicious cycle leading to mitochondrial damage and neuronal cell death in epilepsy.

Acknowledgement. This study was supported by grants of the University of Bonn (BONFOR) and of the Deutsche Forschungsgemeinschaft (Ku 911/ 11-1) to WSK.

References:
1. Kudin AP, Kudina TA, Seyfried J, Vielhaber S, Beck H, Elger CE, Kunz WS. Seizuredependent modulation of mitochondrial oxidative phosphorylation in rat hippocampus. Eur J Neurosci. (2002) 15:1105-14.
2. Kunz WS, Kudin AP, Vielhaber S, Blumcke I, Zuschratter W, Schramm J, Beck H, Elger CE. Mitochondrial complex I deficiency in the epileptic focus of patients with temporal lobe epilepsy Ann Neurol (2000) 48:766-73.
3. Kunz WS, Goussakov IV, Beck H, Elger CE. Altered mitochondrial oxidative phosphorylation in hippocampal slices of kainate-treated rats. Brain Res. (1999) 826:236-42.

In experimental animal models and in human surgical tissue from intractable epileptic patients, there is often sclerosis of the hippocampus that may exacerbate the patient’s seizures. A better understanding of how damage is initiated, how it progresses and what intrinsic neuroprotective mechanisms may be at play, are necessary if we are to develop new therapies to counteract intractable epilepsy.
Using organotypic hippocampal slice cultures and acutely prepared hippocampal slices, we have combined a model of interictal type epileptiform activity with semi quantitative imaging and RT-PCR to identify the extent of epilepsy induced damage and immediate early gene transcription. Our aim is to understand how mild epileptiform activity can initiate immediate early gene expression that either damages or protects the hippocampal neurones.
Our model of focal epilepsy uses bicuculline, a GABAA receptor antagonist, that elicits short regular interictal type epileptiform activity in hippocampal subregions. To determine the extent of damage elicited within this model we have compared the damage it causes with that caused by two other agents reported to induce seizure-like activity: 3-nitropropionic acid, an inhibitor of succinate dehydrogenase (3-NPA); and kainic acid (KA), a known excitotoxin. To determine the extent of the damage caused, slices were exposed to the three different agents for 4hrs, followed by 15hrs recovery. A dual-stain fluorescent assay assessed viable (calcein-AM, 4 mM) and damaged cells (propidium iodide, 20 mM). Confocal images were collected (BioRad) from four randomly selected areas within hippocampal subregions CA1 and CA3, using a 60x water-immersion objective. Fluorescence area was determined using Simple-PCI (C-Imaging). Electrophysiological field potential recordings determined, in separate slices, the extent of epileptiform activity under the three conditions. We have also determined in separate experiments, using RT-PCR, the activation by the mild form of bicuculline induced epileptiform activity of a number of potential immediate early gene candidates.
The results show how the damage caused by interictal type epileptiform activity in the hippocampus is very mild in comparison with the other models, but in all models the damage starts early and progresses. The discussion will explore some of our results that support the idea that ongoing interictal type activity may actually be protective, perhaps via activation of the novel immediate early genes we have identified.

Acknowledgement. We acknowledge the support of the Epilepsy Research Foundation UK and Action Research UK.

References:
1. Empson RM, Jefferys JG. Ca2+ entry through L-type Ca2+ channels helps terminate epileptiform activity by activation of a Ca2+ dependent afterhyperpolarisation in hippocampal CA3. Neuroscience. (2001) 102:297-306.
2. Empson RM, Gee VJ, Sheardown MJ, Newberry NR. Chlormethiazole inhibits epileptiform activity by potentiating GABA(A) receptor function. Brain Res. (2000) 884:31-4.
3. Schmitz D, Empson RM, Gloveli T, Heinemann U. Serotonin blocks different patterns of low Mg2+-induced epileptiform activity in rat entorhinal cortex, but not hippocampus. Neuroscience. 76:449-58 (1997)

Seizure-like events (SLE) induced by low-[Mg²⁺] ACSF perfusion of transverse hippocampal slices from young rats were characterised by a wide range of frequencies from <1 Hz up to 800 Hz. Wavelet transform of CA3 field potential recordings, disclosed a single rhythm declining from about 200 Hz at the onset of SLE to below 1 Hz in the clonic phase with a discontinuity at the tonic-clonic border [1]. SLEs were preceded by a few paroxysmal spikes (PS). These, along with the onset of SLE frequently started with a fast negative deflection increasing in amplitude as approaching the SLE onset. Both PSs and SLE onsets were associated with high frequency oscillations (HFO, > 300 Hz), that often shown up at the initiation of these events, disappeared in the tonic phase and reappeared in the clonic phase. HFO energy dropped at the SLE onset and remained low during the clonic phase. Single CA3 neurones expressed action potential firing at substantially lower frequencies (40-160 Hz). Composite postsynaptic currents, associated with PSs and SLE onsets, arose from a flat baseline and frequently showed a smooth rising phase of 2-10 ms. The risetime of composite postsynaptic currents associated with SLE discharges shoved that the synchrony increased with PSs, peaked around the SLE onset, decreased with the evolution of tonic phase discharges, and after a slight increase, remained stable for the clonic phase. These data indicate, that multiple subgroups of synchronously firing neuronal aggregates coexist, and the number of these decreases, while the size increases as the system approaches the SLE onset.

Acknowledgement. This work was supported by grants 1/047 NKFP MediChem, OTKA T 035225, (Hungary), and the Center of Excellence on Biomolecular Chemistry (Project CBCH) QLK2-CT-2002-90436.

References:
1. Nyikos, L., Lasztóczi, B., Antal, K., Kovács, R., and Kardos, J. Desynchronisation of spontaneously recurrent experimental seizures proceeds with a single rhythm. Neurosci. (2003) 121:705-717.
2. Lasztoczi B, Kovacs R, Nyikos L, Kardos J. A glutamate receptor subtype antagonist inhibits seizures in rat hippocampal slices. Neuroreport. (2002), 13:351-6

Pathological synchronisation is generally believed to be associated with epileptiform activity. Due to the nonstationary and noisy character of signals, however, traditional Fourier-based spectral and coherence methods are not well suited to study epileptiform activity, because they assume stationarity. In contrast, complex wavelet transform (CWT) methods are suitable for nonstationary signals and offer the additional advantage of fine-tuning the analysis by selecting the best wavelet from a number of wavelet families. Wavelets were shown to be useful to characterise the instant frequency components in seizure like events (SLE) induced by low extracellular [Mg²⁺] in transversal hippocampal slices from young rats [1]. To follow the temporal variations in synchrony of extra- and intracellular signals, the Hilbert transform (HT) method was shown [2] to be suitable to quantify time-dependent phase locking of field potential and either membrane potential or membrane current recorded during and between SLEs. In addition to the HT method, we also use the CWT method to calculate phase synchrony during interictal activity and SLEs (Fig. 1). The two methods are theoretically equivalent, but CWT is preferred in practice since bandpass filtering required by the HT method is unnecessary. Between 0.25-2 Hz, the mean phase coherence is found to be high in the interictal period (not shown) with occasional losses of coherence as the SLE approaches – a phenomena which might be utilised for seizure prediction or anticipation.

Fig. 1. Mean phase coherence (R, vertical axis), calculated by the CWT method, of the field potential and the membrane potential for two consecutive SLEs recorded in the low- [Mg2+] model, as a function of time (t) and frequency (w). The R values are close to unity during the SLEs, with a transient decoherence period in the tonic phase appearing at lower frequencies.

Acknowledgement. This work was supported by grants 1/ 047 NKFP MediChem, OTKA T 035225, (Hungary), and the Center of Excellence on Biomolecular Chemistry (Project CBCH) QLK2-CT-2002-90436.

References:
1. Nyikos, L., Lasztóczi, B., Antal, K., Kovács, R., and Kardos, J. Desynchronisation of spontaneously recurrent experimental seizures proceeds with a single rhythm. Neurosci. (2003) 121:705-717.
2. Lasztóczi, B., Nyikos, L., Antal, K., Emri, Zs. and Kardos, J. Synaptic transmission shapes dynamics of nonlinearly coupled neuronal oscillators in low-[Mg2+] induced seizures. Eur. J. Neurophysiol. (2004) 19:1361-1372.