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Grenzflächen verbinden Räume, die gleichzeitig unterschiedlich und genügend ähnlich sein müssen. Dabei steuern sie Quantität sowie Qualität eines über sie stattfindenden Austauschs. Grenzflächen sind in den Dimensionen des Raums und der Zeit hochkomplex, selbstorganisiert, von endlicher Ausdehnung und tolerieren nur geringe Veränderungen des Milieus. Die Grenzflächen-Funktion ist ein aktiver Prozess, der Austausch ermöglicht, Energie von außen braucht, hochspezifisch ist, Entropie verringert und Information vergrößert und auf diese Weise Unterschiedlichkeit herstellt und aufrechterhält. Wenn auch nur eine der Eigenschaften der Grenzfläche ausfällt, bricht das System zusammen. Die Grenzflächen-Funktion ist ein physiologisches Grundprinzip, das hier verwendet wird, um die Hypothese einer analogen Grundfunktion im Psychischen aufzustellen. Grenzflächen können in Analogie zur Kontaktschranke im Bion’schen Sinn gedacht werden, sie finden sich ubiquitär und funktionieren simultan. Diese Überlegungen werden anhand von klinischen Fallbeispielen aus psychoanalytischen Behandlungen veranschaulicht. Dabei werden verschiedene Eigenschaften der Grenzflächenfunktionen sowie Aspekte ihrer Störung, respektive Wiederherstellung beleuchtet.

Pentylenetetrazole (PTZ) is a convulsant used to model epileptic seizures in rats. In the PTZ-model, altered heat shock protein 27 (HSP-27) expression highlights seizure-affected astrocytes, which play an important role in glutamate and GABA metabolism. This raises the question whether impaired neurotransmitter metabolism leads to an imbalance in neurotransmitter receptor expression. Consequently, we investigated the effects of seizures on the densities of seven different neurotransmitter receptors in rats which were repeatedly treated with PTZ (40 mg/kg) over a period of 14 days. Quantitative in vitro receptor autoradiography was used to measure the regional binding site densities of the glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA) receptors, the adenosine receptor type 1 (A(1)), which is part of the system controlling glutamate release, and the gamma-aminobutyric acid (GABA) receptors GABA(A) and GABA(B) as well as the GABA(A)-associated benzodiazepine (BZ) binding sites in each rat. Our results demonstrate altered receptor densities in brain regions of PTZ-treated animals, including the HSP-27 expressing foci (i.e. amygdala, piriform and entorhinal cortex, dentate gyrus). A general decrease of kainate receptor densities was observed together with an increase of NMDA binding sites in the hippocampus, the somatosensory, piriform and the entorhinal cortices. Furthermore, A(1) binding sites were decreased in the amygdala and hippocampal CA1 region (CA1), while BZ binding sites were increased in the dentate gyrus and CA1. Our data demonstrate the impact of PTZ induced seizures on the densities of kainate, NMDA, A(1) and BZ binding sites in epileptic brain. These changes are not restricted to regions showing glial impairment. Thus, an altered balance between different excitatory (NMDA) and modulatory receptors (A(1), BZ binding sites, kainate) shows a much wider regional distribution than that of glial HSP-27 expression, indicating that receptor changes are not following the glial stress responses, but may precede the HSP-27 expression.

A-type currents powerfully modulate discharge behavior and have been described in a large number of different species and cell types. However, data on A-type currents in human brain tissue are scarce. Here we have examined the properties of a fast transient outward current in acutely dissociated human neocortical neurons from the temporal lobe of epilepsy patients by using the whole-cell voltage-clamp technique. The A-type current was isolated with a subtraction protocol. In addition, delayed potassium currents were reduced pharmacologically with 10 mM tetraethylammonium chloride. The current displayed an activation threshold of about -70 mV. The voltage-dependent activation was fitted with a Boltzmann function, with a half-maximal conductance at -14.8 +/- 1.8 mV (n = 5) and a slope factor of 17.0 +/- 0.5 mV (n = 5). The voltage of half-maximal steady-state inactivation was -98.9 +/- 8.3 mV (n = 5), with a slope factor of -6.6 +/- 1.9 mV (n = 5). Recovery from inactivation could be fitted monoexponentially with a time constant of 18.2 +/- 7.5 msec (n = 5). At a command potential of +30 mV, application of 5 mM 4-aminopyridine or 100 microM flecainide resulted in a reduction of A-type current amplitude by 35% or 22%, respectively. In addition, flecainide markedly accelerated inactivation. Current amplitude was reduced by 31% with application of 500 microM cadmium. All drug effects were reversible. In conclusion, neocortical neurons from epilepsy patients express an A-type current with properties similar to those described for animal tissues.

Spreading depression (SD) is characterized by a transient breakdown of neuronal function concomitant with a massive failure of ion homeostasis. It is a phenomenon that can be induced in neocortical tissue by raising excitability, e.g. injection of K(+), application of glutamatergic agonists, or blocking Na(+)/K(+) ATPase. Here we report a novel method of SD induction using minimal disinhibition with application of low concentrations (5 microM) of the GABA(A) receptor blocker bicuculline. This procedure-while subthreshold for epileptiform activity-readily induced spontaneous SDs in native rat neocortical slices, accompanied by typical depolarizations of neurons and glial cells. In contrast, in human neocortical preparations obtained from epilepsy surgery, in approximately 20% of the slices spontaneous epileptiform activity appeared with this bicuculline dosage without SDs. Raising the concentration of bicuculline to an epileptogenic dose (10 microM) in human tissue also resulted in the generation of epileptiform activity only. Likewise, in slices from pilocarpine-treated, chronically epileptic rats, bicuculline also only induced epileptiform activity without eliciting SDs. The experiments indicate that chronic epilepsy causes a differential sensitivity to partial GABA(A) receptor blockade with regard to induction of SD.

Intra- and extracellular recording techniques were used to study the epileptiform activity generated by guinea pig hippocampal slices perfused with free-magnesium artificial cerebrospinal fluid in the presence of physiologic (4 mM), reduced (2 mM) or elevated (8 mM) extracellular potassium concentrations ([K+]o). Extracellular field potentials along with intracellular recordings were recorded in CA1 or CA3 region. Reduction of [K+]o significantly increased the latency of epileptiform field potential (EFP) appearance as well as burst discharge duration and decreased EFP repetition rate. Depending on different background [K+]o, epileptiform burst discharges appeared in different patterns including varied types of paroxysmal depolarisation shifts and burst activity in CA1 and CA3 subfields. Comparison with physiological and increased [K+]o, reduction of [K+]o significantly increased the mean duration of bursts, mean amplitude of depolarisation, mean after-hyperpolarisation duration, and inter-spike intervals in both CA1 and CA3 areas. Three distinct patterns were distinguished on the basis of their evoked firing pattern in response to application of depolarising current pulses in the interval of epileptiform burst discharges. Neurons superfused with 2 mM [K+]o presented fast adapting pattern while cells washed with 4 or 8 mM [K+]o exhibited intrinsically bursting or slow adapting patterns. Comparing the groups with different background [K+]o, there is a more severe form of discharges in low K+ and a subtle difference between 4 and 8 mM K+. The data indicate the importance of background [K+]o on epileptiform burst discharge pattern and characteristics.

Summary: Purpose: The antiepileptic drug vigabatrin (VGB) increases intracellular availability of the inhibitory transmitter γ-aminobutyric acid (GABA) by inhibition of GABA-transaminase. A blockade of the GABA uptake is the main mechanism of action of tiagabine (TGB). Based on this, the two antiepileptic drugs (AEDs) can be speculated to act synergistically so that their combined antiepileptic efficacy is supraadditive. Methods: To test this, experiments were performed on hippocampal slices of guinea-pigs. As an epilepsy model, epileptiform field potentials (EFPs) were induced by omission of Mg2+ from the bath solution and recorded in stratum pyramidale of the CA3 region. VGB (7.5 μM) and TGB (0.75 μM) were added to the superfusate. Results: VGB, given alone, failed to decrease the repetition rate of EFPs. Similarly, TGB applied alone only transiently led to a nonsignificant reduction of the EFP frequency. Combining VGB and TGB, their suppressive efficacy increased, yielding a significant reduction of EFP frequency, which, however, again did not persist. Pretreatment of the preparations with VGB for 2 h, followed by additional application of TGB, or TGB alone, drastically and persistently potentiated the effects. Conclusions: These results demonstrate that VGB and TGB show favorable pharmacodynamic interactions, provided VGB is allowed to block intracellular GABA degradation before GABA uptake block by TGB.

Summary: Purpose: The anticonvulsant effects of the novel antiepileptic drug (AED) levetiracetam (LEV) were tested in neocortical slice preparations from 23 patients who underwent surgery for the treatment of refractory epilepsy. Methods: Slices were used to evaluate the effects of LEV on two different models of epilepsy: low-Mg2+–induced untriggered and bicuculline-evoked stimulus-triggered epileptiform burst discharges and spontaneously appearing rhythmic sharp waves. Results: LEV (0.1–1 mM) did not influence spontaneously appearing rhythmic sharp waves or Mg2+-free aCSF-induced epileptiform field potentials. LEV affected neither the amplitudes or duration nor the repetition rates of burst discharges in these epilepsy models. However, LEV (100–500 μM) significantly suppressed the ictal-like discharges elicited by the γ-aminobutyric acid subtype A (GABAA)-receptor antagonist bicuculline. A marked reduction of the amplitude and duration of bicuculline-evoked field response in the presence of LEV was observed. Conclusions: The results indicate the potential for LEV to inhibit epileptiform burst discharges in human neocortical tissue, which is consistent with its effects in animal models of epilepsy. These results also support the seizure reduction observed in clinical trials and support that this may, in part, be related to the ability of LEV to inhibit epileptiform discharges.

Cyclosporine A (CsA) neurotoxicity is a common cause of seizures in transplant patients and others receiving immunosuppressive therapy. CsA at concentrations higher than the levels estimated for cerebrospinal fluid of the patients suffering from seizure attacks was ineffective to induce epileptiform field potentials (EFP) in in vitro brain-slice preparation. The aim of this study was to test the effect of CsA at lower concentrations on neuronal activity. Guinea-pig hippocampal slices were exposed to artificial cerebrospinal fluid containing CsA (0.1-2 microM). Furthermore, the effects of CsA (0.25-10 microM) were tested on EFP elicited by omission of Mg2+ from superfusate. Low concentrations of CsA (0.1-0.25 microM) induced EFP while higher doses (0.5-2 microM) failed to decrease the seizure threshold. CsA at concentrations of 0.25 and 1 microM had no significant effect on the low Mg2+-induced EFP. Higher CsA concentration (10 microM) strongly suppressed EFP. The results indicate that CsA at doses that are probably clinically relevant increases the neuronal excitability.

Eine der wesentlichen Fragen bei der Untersuchung chronisch epileptischen Hirngewebes ist die, ob dieses Gewebe, welches im Patienten epileptische Entladungen, also synchrone Netzwerkaktivitat gezeigt hat, dies auch in vitro beibehalt. Um dieses Problem anzugehen, wurden bioelektrische Registrierungen in menschlichen Schnittpraparaten des Temporal- bzw. Frontallappens aus epilepsiechirurgischen Eingriffen mit konventionellen intra- und extrazellularen Registriertechniken sowie mit optischen Methoden (spannungsempfindliche Farbstoffe) vorgenommen. Es konnte gezeigt werden, dass menschliches epileptisches Gewebe tatsachlich spontane scharfe Wellen in vitro aufweist, denen auf zellularer Ebene polyphasische, vorwiegend hyperpolarisierende postsynaptische Potenziale entsprechen. Diese beruhen sowohl auf exzitatorischen wie inhibitorischen Prozessen. Das neuronale Netzwerk zur Generierung von Summenaktionspotenzialen ist offenbar sehr klein. Die spontanen Ereignisse beginnen meist in supragranularen Schichten, haufig mit multiplen Foki. Die Synchronisation neuronaler Depolarisationen innerhalb der initiierenden Foki ist gering, wie mit Hilfe von spannungssensitiven Farbstoffen gezeigt werden konnte. Bei experimenteller Steigerung der Epileptogenitat wird iktaforme Aktivitat ebenfalls in kleinen Foki mit einem Durchmesser von 100 - 300 µm initiiert, um dann in eine extreme neuronale Synchronisation uberzugehen. Die Untersuchungen weisen darauf hin, dass multiple spontan aktive Foki in chronisch epileptischem Hirngewebe vorliegen und dass Anfalle durch eine Synchronisierung dieser Foki entstehen. One of the crucial questions when investigating chronically epileptic human resectates is whether this tissue, which in patients did generate synchronous network activity in the way of epileptiform discharges, will continue to do so also in vitro. To address this issue, bioelectrical recordings were done in human temporal and frontal lobe slices resected from therapy-resistant patients during epilepsy surgery using conventional extra- and intracellular recording techniques as well as voltage-sensitive dyes. It could be shown that human epileptic tissue indeed displays spontaneous sharp waves in vitro, which on the intracellular level are reflected by polyphasic, mainly hyperpolarizing postsynaptic potentials. These are based on both excitatory and inhibitory synaptic processes. Grouped action potentials were observed as populations spikes; however, the neuronal networks required to generate these discharges appear to be very small. Initiation of epileptic discharges often occurs in supragranular layers with multiple foci. The degree of synchronisation within the initiatory focus is low, as revealed by voltage-sensitve imaging. With an experimental increase in epileptogenicity, ictaform activity with massive synchronization is also initiated in small foci of 100 - 300 µm diameter. It is concluded that multiple, spontaneously active foci exist in chronically epileptic tissue and that seizures may result from a synchronisation of these foci.

Dystonic mutant dt(sz) hamsters are a model for paroxysmal dystonia. Handling/stress provoke the dystonic attacks. This phenomenon subsedes with maturation, but can be reinvoked when these animals receive sodium channel blockers such as lamotrigine, suggesting a dysfunction of striatal sodium channels. Voltage-gated fast sodium currents (I(Na(+))) were studied in acutely isolated striatal neurons from healthy and dt(sz) hamsters in whole-cell voltage clamp recordings. The action of lamotrigine was tested on (a) current/voltage relationship, (b) kinetics, and (c) steady-state inactivation and activation. Under control conditions, properties of I(Na(+)) were not different between healthy and dt(sz) neurons. With lamotrigine, however, (a) peak currents were significantly less depressed by the drug in neurons from dt(sz) hamsters as compared to healthy cells, and (b) the steady-state inactivation curve shift of I(Na(+)) was less pronounced in dt(sz) neurons. The results suggest that in dt(sz) hamsters, fast sodium currents in striatal neurons are more resistant to blockade. This sodium channel alteration might be causal for a functional imbalance between input and output structures of the basal ganglia under conditions of compromised I(+)(Na).

The antiepileptic effects of the novel antiepileptic drug retigabine (D-23129) [N-(2-amino-4-(4-flurobenzylamino)phenyl) carbamid acid ethyl ester] were tested in neocortical slice preparations (n=23) from 17 patients (age, 3-42 years) who underwent surgery for the treatment of intractable epilepsy. Epileptiform events consisted of spontaneously occurring rhythmic sharp waves, as well as of epileptiform field potentials (EFP) elicited by superfusion with Mg(2+)-free solution without or with addition of 10 micromol/l bicuculline. (1) Spontaneous rhythmic sharp waves (n=6), with retigabine application, the repetition rate was decreased down to 12-47% of initial value (10 micromol/l, n=3) after 180 min or suppressed completely within 12 min (50 micromol/l, n=3). (2) Low Mg(2+) EFP (n=9), with retigabine application, the repetition rate was decreased down to 50 and 65% of initial value (10 micromol/l; n=2) after 180 min or suppressed completely after 9-55 min (10, 50 and 100 micromol/l; n=2 in each case). In one slice only a transient reduction of the repetition rate was seen with 10 micromol/l retigabine. (3) Low Mg(2+) EFP with addition of bicuculline (n=8), with retigabine application, the repetition rate was decreased down to 12-55% of initial value (10 micromol/l; n=4) after 180 min or suppressed completely after 6-30 min (50 and 100 micromol/l; n=2 in each case). The depressive effect of retigabine was reversible in all but one slice. The results show a clear antiepileptic effect of retigabine in human neocortical slices on spontaneously occurring rhythmic sharp waves and different types of induced seizure activity.

Living brain slices are usually cut with razor blades, which compress a ca. 50-microm-thick layer of tissue. This results in cell debris and lesioned cells which, e.g. form diffusion barriers between the bath and living neurons underneath, thereby prolonging response times of neurons to drugs in the bath saline and impeding the experimental access to intact neurons. To avoid such drawbacks, a macromingotome was developed which cuts nervous tissue with water jets. Physiological saline under pressures of 100-1800 bar was ejected through nozzles of 35-100 microm to cut 300-500-microm-thick hippocampal slices. Systematic variations of pressure and nozzle diameter revealed best results at 400-600 bar and with nozzle diameters of 60-80 microm. Under these conditions, intact CA1- and CA3-neurons as well as granule cells were detected with infrared microscopy at less than 10 microm underneath the surface of the slice. Superficial neurons with intact fine structures were also seen when the slices were studied by light-microscopy. Intra- and extracellular recordings from superficial neurons showed normal membrane- and full action potentials and the development of stable epileptiform discharges in 0 Mg(2+)-saline. These results indicate that the macromingotome offers an alternative way of cutting slices which may facilitate electrophysiological/neuropharmacological or fluorometric studies on superficial neurons.

Spreading depressions (SD) occur in association with ischaemia, epilepsy and migraine. Intracellular calcium oscillations have been suggested to be involved in the generation and propagation of SD. The present study was performed to study the mechanism of conditioning guinea pig hippocampal slices by the T-type calcium channel blockers NiCl2 and amiloride. SD-like fluctuations of DC potential were recorded by inserting microelectrodes into the CA1 and CA3 regions. The SD occurrence was significantly greater with 10 µmol/ l NiCl2 as well as with 25 and 50 µmol/ l amiloride than with other concentrations of these substances. The concentration response curve was inversely U-shaped with the maximum repetition rates of SDs being achieved at 10 µmol/ l NiCl2 as well as at 25 and 50 µmol/ l amiloride. SD occurrence could be completely blocked by the NMDA antagonist APV (10 µmol/ l) in all cases. These data demonstrate that modulation of the Ca2+ dynamics conditioned guinea pig hippocampal slices and increased their susceptibility to generate SD.

The structure effect relationships of derivatives of the antiepileptically active ester of valproate (VPA) 3,4:5,6-Di-O-isopropylidene-1-O-(2-propylpentanoyl)- d -mannitol (1) have been studied using intracellular recording to record the membrane potential of single neurons (buccal ganglia, Helix pomatia). Epileptiform activity was induced by the epileptogenic drug pentylenetetrazol. The effects of several derivatives on epileptiform activity were compared with those of the relay compound 1. Most of the synthesized agents decreased the duration of paroxysmal depolarization shifts (PDS) and increased their repetition rate. It was considered that a decreased the duration of PDS is antiepileptic and an increased repetition rate is pro-epileptic. Compared with the effects of compound 1, the following relationships were found: (1) Derivatives containing glucitol or galactitol were of similar antiepileptic potency. (2) Introduction of pyranoses or furanoses rendered the substances inactive or even pro-epileptic. (3) VPA in position 1 and 6 at the sugar acted as an antiepileptic whereas in position 3 and 4 it proved to be ineffective. (4) Replacement of VPA by ethylhexanoyl reduced the antiepileptic potency slightly and pivaloyl strongly. (5) Replacement of isopropylidene bridges by penta-O-acetyl or cyclohexylidene residues led to largely inactive substances. (6) Compounds having isopropylidene bridges in position 2,4;3,5 proved to be antiepileptic whereas bridges especially in positions 2,3:4,5 slightly enhanced epileptic activities.

Derivatives of the antiepileptic drug valproate (VPA, 2-propylpentanoic acid) have been synthesized and tested in order to improve the intracellular availability of VPA. The buccal ganglia of Helix pomatia were used as a test nervous system and antiepileptic efficacies were reconfirmed using rat cortex in vivo. Epileptiform activities consisted of typical paroxysmal depolarization shifts (PDS) which appeared in the identified neuron B3 with application of pentylenetetrazol. Epileptiform activities were found to be accelerated, unaffected or blocked. (i) The Amide-derivatives 2-propylpentanamide and N,N-dipropyl-2-propylpentanamide, and short chain ester derivatives 1-O-(2-propylpentanoyl)-2,3-propandiol, 2,2-di(hydroxymethyl)-1-O-(2-propylpentanoyl)-1,3-propanediol and 2,2-di(hydroxymethyl)-1,3-di-O-(2-propylpentanoyl)-1,3-propanediol accelerated epileptiform activities. Membrane potential often shifted to a permanent depolarization which corresponded to the PDS-inactivation level. (ii) The structural analogs 1-cycloheptene-1-carboxylic acid and cyclooctanecarboxylic acid accelerated epileptiform activities only slightly or were without effects. (iii) The small VPA-ester, 2-propylpentanoic acid ethyl ester, decreased the epileptiform activities in a way that is comparable to the effects of VPA well known from previous studies. It thus could be thought as a VPA-pro-drug. (iv) The mannitol-esters 1-O-(2-propylpentanoyl)-D-mannitol and 3,4;5,6-Di-O-isopropylidene-1-O-(2-propylpentanoyl)-D-mannitol blocked the PDS in a way which is different from the known effects of VPA. These substances are interpreted not to exert their effects after being metabolized to VPA and thus they are thought to be new antiepileptic substances.

Since a disturbed balance between excitatory and inhibitory amino acid receptors is suggested to be an important condition for epileptogenic cortical activity, the present study has focused on the analysis of the densities of (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-D-aspartate, kainate and GABA subtype A receptors in neocortical tissue surgically removed from patients with focal epilepsy. The mean densities (collapsed over cortical layers I-VI) and the laminar distribution patterns of [3H]AMPA, [3H]MK-801, [3H]kainate and [3H]muscimol binding to AMPA, N-methyl-D-aspartate, kainate and GABAA receptors were determined with quantitative receptor autoradiography in the neocortex of patients with focal epilepsy and controls. The tissue probes used in the present study were functionally characterized by parallel electrophysiological investigations. From that, the different probes could be subdivided into a spontaneously spiking and a non-spontaneously spiking group. The mean density of [3H]AMPA binding sites was significantly increased (+37%) in the group of epileptic brains (n = 10) compared with controls (n = 10), but the mean densities of [3H]MK-801, [3H]kainate and [3H]muscimol binding sites were not significantly altered (-8%, +/-0% and -7%, respectively). The relation between the densities of all four binding sites were simultaneously displayed as polar plots in each single brain ("receptor fingerprints"). The consistent up-regulation of [3H]AMPA binding sites in all epileptic brains was found to be associated with a down-regulation of the N-methyl-D-aspartate receptor in four of the five non-spontaneously spiking cases, and an associated up-regulation of the N-methyl-D-aspartate receptor was seen in all spontaneously spiking cases. Finally, the laminar distribution of binding site densities was analysed, since the mean densities collapsed over all neocortical layers may obscure layer-specific alterations. Layer- and receptor- specific up- or down-regulations were found in epileptic tissue compared with controls. Moreover, the laminar distribution pattern of current sinks associated with epileptiform potentials in a spontaneously spiking cortical slice was found to be co-localized with local maxima of AMPA receptor densities. The present analysis of four ionotropic glutamate and GABA receptor subtypes demonstrates a consistent and significant up-regulation of [3H]AMPA binding sites in all cases of human focal epilepsy, which co-localizes with the occurrence of sinks in current-source-density analysis. The receptor fingerprint analysis suggests a subdivision of focal epilepsy into two subtypes on the basis of neurochemical/functional correlations: (i) a spontaneously spiking subtype with increased N-methyl-D-aspartate receptor density, and (ii) a non-spontaneously spiking subtype with decreased N-methyl-D-aspartate receptor density.

In human neocortical slices obtained during epilepsy surgery, sharp waves have been described to appear spontaneously, the shape of which met all criteria of epileptiform field potentials. In the present investigation, the current sinks and sources underlying these potentials were analysed. The cortical tissue used in the present study was a small portion of the tissue blocks excised for treatment of pharmacoresistant focal epilepsy. The tissue came from the temporal (n = 26), frontal (n = 1) and parietal (n = 1) lobes. Slices of 500 microm thickness were cut in the frontal plane perpendicular to the pial surface. Field potentials were recorded using a linear array of eight wire electrodes (diameter: 33 microm) with interelectrode distances of 300 microm. To scan a slice for sharp field potentials, this array was placed perpendicular to the pial surface at the midsection of each preparation, and consecutively at the respective midsections of the resulting halves of the slice. Each of these locations was termed a recording line. Depending on the appearance of spontaneous potentials, recording lines and slices were classified as either spontaneous or non-spontaneous. With both spontaneous and zero Mg(2+)-induced interictal discharges, in spontaneous slices, current sinks were preferentially located in layers II and III. In non-spontaneous slices, current sinks associated with interictal potentials could be found throughout all cortical laminae. With zero Mg(2+)-induced ictal activity, in spontaneous slices, the initial sinks were preferentially located in cortical laminae II and IIIa, and were shifted to lower ones after additional application of bicuculline. In non-spontaneous slices, no ictal-type discharges could be induced with omission of Mg2+ from the superfusate. Only addition of bicuculline elicited ictal-type activity, and sinks associated with this were preferentially located in layers II and IIIa. The results suggest that the supragranular layers, especially layer II, change qualitatively in functional organization in slices showing spontaneous discharges. We think that this special feature represents the function of the upper layers and can be blocked by bicuculline. This interpretation is supported by the observation that ictal discharges normally started in the upper layers in spontaneous and non-spontaneous slices, except for spontaneous slices with bicuculline, where the zone initiating discharges was translocated to deeper layers.

The aim of the present investigation was to compare the antiepileptic efficacy of the specific L-type calcium channel blocker nifedipine in hippocampal and neocortical slice preparations in the Mg2+-free model of epilepsy. The main findings were as follows. (1) In hippocampal slices, in general, nifedipine (20-80 micromol/l) exerted a suppressive effect both on repetition rate and on area under epileptiform field potentials. This effect was clearly dose dependent. In the majority of cases, this suppression was preceded by an increase, which was transient in nature. Only in the lowest concentration (20 micromol/l) used, in normal K+, instead of a depression, a persistent increase occurred. (2) In neocortical slices, in the majority of experiments, nifedipine (20-80 micromol/l) showed a depressive action only on the area under the epileptiform field potentials. The depressive effect of nifedipine on the area was dose dependent, although to a lesser extent than in the hippocampus. In nearly half of the slices this suppression was preceded by a transient increase. By contrast, the repetition rate of epileptiform field potentials increased transiently in about 20% of the slices followed by a decrease. In the remaining 80% of the slices the repetition rate increased persistently. (3) An elevation of the K+ concentration accentuated the depressive actions of nifedipine only in the hippocampus. In contrast to elevated K+, in both the hippocampus and the neocortex, epileptiform field potentials were not suppressed in all experiments in normal K+. (4) The reversibility of the depressive effects of nifedipine was differential in the two tissue types. In the hippocampus, after suppression of epileptiform field potentials they reappeared in the overwhelming majority of slices. In the neocortex, this was the case in only one experiment. These findings may indicate the existence of L-type calcium channels with a differential functional significance for epileptogenesis and/or the existence of different forms of L-type channels in hippocampal and neocortical tissue. As a whole, the differential effects of L-type calcium channel blockade in the hippocampus and neocortex point to differences in the network properties of the two tissue types.

The actions were examined of 17 frequently used glycol ether compounds on the glutamate receptor-mediated ion currents. The receptors were expressed in Xenopus oocytes by injection of rat brain mRNA. Most of the 17 glycol ethers exerted no effects on the glutamate subreceptors activated by kainate and N-methyl-D-aspartate (NMDA), whereas 2-phenoxyethanol (ethylene glycol monophenyl ether) caused a considerable reduction of NMDA-induced membrane currents in a reversible and concentration-dependent manner. The threshold concentration of the ethylene glycol monophenyl ether effect was10 mumol/l. The concentration for a 50% inhibition (IC50) was approximately 360 mumol/l. The results indicate a neurotoxic potential for 2-phenoxyethanol.

For investigations of hypoxic effects in nervous tissue, brain slices are often used as a model system. This provides the advantage that parameters of the micromilieu, e.g. pH and temperature can easily be controlled and measurements of different data, e.g. bioelectric potentials, ion activities etc. can be performed. It is of special importance that the PO2 the slice preparation is exposed to is equally controlled under these conditions. Therefore, a PO2 monitoring system is needed which provides representative values for the tissue environment. This requirement is fulfilled by an optical PO2 sensing method based on phosphorescence quenching as a function of PO2. Here, the application of this method as adapted for use in in vitro models is described and compared to the polarographic oxygen-sensing method. Both the optical and polarographic methods are comparable regarding accuracy and response time of measurements. Furthermore, both the optical method and electrophysiological measurements can be combined. Lastly, under experimental conditions, neither the phosphorescent dye Palladium-meso-tetra-4-carboxyphenyl-porphine nor the illumination necessary for excitation of the dye influence bioelectric activity of neuronal tissue in vitro. In conclusion, the optical PO2 sensing method presented here provides a tool for reliable and continuous monitoring of PO2 in the immediate environment of brain slice preparations.

Up to now microsurgical dissections in living nervous tissue (e.g. in slices or cell cultures) are performed either by micro-scalpels or by laser beams. As an alternative technique, a device for cutting with an ultrafine pulsed water jet was developed to allow precise, visually controled dissections in neuronal circuits even during electrophysiological recordings. Water is ejected by pressure (20–30 bar) from patch pipettes with tip diameters of 10–12 μ m. By means of a piezo-element the pipette and the water jet are forced to oscillate vertically with a frequency of 200–400 Hz with an adjustable amplitude. These oscillations facilitate the transsection of neuronal connections even in thick slice preparations. Best results were obtained when the tip of the pipette was about 500 μ m above the surface of the submerged slice tissue. This micromingotome offers the following advantages: (i) histological studies show that the water jet cleans the cutting surface, thus avoiding debris and its uncontrolable effects on cells underneath; (ii) the arrangement enables ongoing electrophysiological recordings from hippocampal slices during the cutting procedure and thus facilitates studies of the functions of neuronal connections; (iii) the device allows even disconnection in cultured nervous tissue overgrowing polyamid grids with 50 μ m wide meshes.

Human neocortical temporal lobe tissue resected for treatment of pharmacoresistant epilepsy was investigated. In slices prepared from this tissue, field potentials sometimes superimposed by population spikes were found to appear spontaneously. In individual slices, they were generalized or highly localized to a field of approximately 200 microns in diameter. Synchronous with these potentials, hyperpolarizing and depolarizing postsynaptic potentials were recorded from neurons in the vicinity of the field potential electrode. Hyperpolarizing postsynaptic potentials appeared to be mainly chloride mediated. All potentials, i.e. sharp field potentials as well as postsynaptic potentials, were reversibly suppressed by blockade of the non-NMDA (non-N-methyl-D-aspartate) glutamate-subreceptor and of the GABAA (gamma-aminobutyric acid) receptor, and by application of the organic calcium channel blocker verapamil. By contrast, all potentials remained unaffected by blockade of the NMDA glutamate-subreceptor and the GABAB receptor. The antiepileptic drugs carbamazepine and phenytoin failed to suppress the spontaneous potentials at therapeutic concentrations. Washout of Mg2+ from the superfusate left the spontaneous potentials unchanged or converted them to ictal-type discharges. This epileptiform activity was not suppressed, but augmented by blockade of the GABAA receptor. As a whole, the spontaneously appearing field potentials may be assumed to reflect a state of increased neuronal synchronization.

1. An enhancement of promoted release of gamma-aminobutyric acid (GABA) and a change in GABA-metabolism have been suggested as mechanisms of action of gabapentin. Vigabatrin is supposed to act mainly via inhibition of GABA-transaminase but it also interferes with GABA-release and GABA-uptake. On the basis of these mechanisms of action, a pharmacodynamic interaction of the two antiepileptic drugs could be supposed which might be of relevance in the sense of a rational polypharmacy. 2. To address the aforementioned hypothesis, experiments were carried out on hippocampal slices (n=107) of guinea-pigs (n=70). Epileptiform field potentials (e.f.p.) were induced by omission of magnesium from the bath solution and recorded in the stratum pyramidale of the CA3 region. Gabapentin (30-600 microM; 5.1-102.72 microg ml(-1)), vigabatrin (50-200 microM, 6.45-25.8 microg ml(-1)) and the GABA(A)-receptor antagonist bicuculline (100 microM) were added to the bath solution for 3 h. 3. Gabapentin, in concentrations up to 600 microM, failed to decrease the repetition rate or duration of e.f.p. (n=19). However, vigabatrin, evoked a dose-dependent reduction of the repetition rate of e.f.p. For a concentration of 100 microM (12.9 microg ml(-1)) there was a reduction down to 48+/-5% (mean+/-s.e.mean) of the initial value within 3 h (n=11). With simultaneous administration of vigabatrin (100 microM) and gabapentin (60 microM) for 3 h (n=15), the repetition rate of e.f.p. decreased down to 8+/-3%, which is significantly different from the values obtained after administration of 100 microM vigabatrin alone (P

The so-called terminal negativity (TN) of the DC-potential is a characteristic reaction of neuronal tissue to hypoxia or ischemia. In a previous study on human neocortical slices, two types of TN with flat and steep slopes of rise (or10 mV/min) were found with hypoxia. The aim of the present study was to further investigate causes underlying the occurrence of flat and steep TN. Experiments were performed on 23 human neocortical slices (500 micron) resected from 13 patients (epilepsy and tumour surgery). DC-potential and evoked potentials (white matter stimulation) were recorded in layer III. The extracellular potassium concentration ([K+]o) was measured by K+-sensitive microelectrodes. In an interface type chamber, ischemic episodes were induced by oxygen and glucose deprivation. They were terminated when TN had peaked. Both flat and steep TN also existed with ischemic conditions. There was a linear correlation between the slope of rise of TN and the associated slope of rise in [K+]o, respectively, but none regarding latencies of TN or recovery of evoked potentials. Peak levels in [K+]o were 13.9+/-0.9 mmol/l. Compared to control, the slope of rise and latency of TN were clearly increased by addition of dimethyl sulfoxide (DMSO, 0.4%) to the bath solution, whereas nimodipine (40 micromol/l) in 0.4% DMSO had neither an effect on slope of rise of TN nor on latency of TN. As a whole, our observations suggest, that the actual metabolic state determines the occurrence of flat or steep TN.

An in vitro human neocortical and rodent hippocampus brain slice technique was used Under repeated hypoxia to investigate the cerebroprotective effect of hypothermia. As a hallmark of the neuronal hypoxic reaction anoxic terminal negativity (ATN) was registered to test whether hypothermia delays the onset of ATN. The experiments clearly confirm an assumed protective effect of hypothermia in vitro and in vivo and give for the first time evidence ofthe lack ofthe protective effect of hypothermia once hypoxia has occurred under normothermic conditions, probably by a critical depletion of cellular ATP-stores. In patients with severe traumatic brain injury and critically low cerebral perfusion pressure mild hypothermia is able to improve clinical outcome. [Neural Res 1998; 20 (Suppl 1): S61-S65]

Alongside GABA, glycine is the major inhibitory transmitter in the central nervous system. Application of the glycine receptor blocker strychnine is known to evoke epileptiform phenomena. The present paper addresses the question whether postsynaptic calcium currents through L-type channels contribute to strychnine-induced epileptiform field potentials (EFP). To test for this, the antiepileptic effect of the specific L-type calcium channel blocker, verapamil, in hippocampal and neocortical slices was investigated. In parallel with this, the antiepileptic efficacy of the unspecific calcium channel modulator, flunarizine, was tested with respect to pharmacotherapy of epilepsies. In both preparations, the L-type calcium channel blocker, verapamil, was able to suppress EFP. In neocortical slices, EFP were blocked in all experiments, whereas in hippocampal slices, in 3 out of 11 experiments, no complete suppression occurred. Flunarizine acted in a similar way. It is concluded that L-type calcium channels are involved in strychnine-induced epilepsy, but to a greater extent in the neocortex than in the hippocampus.

Die gesamte Korpermotorik last sich unter funktionellen Gesichtspunkten in eine Haltungsmotorik und Bewegungsmotorik unterteilen. Ein ungestorter Ablauf der Gesamtmotorik ist nur dann gewahrleistet, wenn die funktionellen Teilbereiche genau aufeinander abgestimmt sind. Ist das nicht der Fall, so treten zwangslaufig Bewegungseinschrankungen, sog. Blockierungen, auf.

The extracellular concentration of glutamate has previously been reported to increase to more than 10-fold the basal level during seizure activity. In the present study, we tested whether localized increases in extracellular glutamate concentration influence the rhythm of epileptiform discharges in the low-magnesium epilepsy model. In hippocampal slices of guinea-pigs, epileptiform activity was induced by omission of magnesium from the bath fluid. Glutamate and its subreceptor agonists N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were ejected into different strata of the CA3 and CA1 regions using microiontophoretic and micropressure application. Glutamate, NMDA and AMPA applied to the CA3 region, but not to the CA1 region, induced a short-lasting increase in epileptiform discharge frequency, often followed by a transient reduction. The effect was most pronounced with application into the stratum lacunosum-moleculare of the CA3 region and could only be evoked in slices exceeding 400 microns in thickness. The effects on the rhythm of epileptiform discharges induced by NMDA and AMPA were blocked by their specific receptor antagonists. They were not influenced by application of GABAA and GABAB receptor antagonists. Changes in somatic membrane potential of CA3 pyramidal neurons did not correlate with changes in the rhythm of epileptiform discharges elicited in this region. The transient suppression of epileptiform discharges that followed the increase in discharge frequency was abolished by an adenosine A1 receptor antagonist. We propose that localized increases in extracellular glutamate concentration modify the rhythm of epileptiform discharges due to changes in neuronal network activity.

In human neocortical slices the specific L-type calcium channel blocker verapamil had been shown to be antiepileptic in the low Mg 2+ -model of epilepsy. The present investigation demonstrated: (1) verapamil exerted also an antiepileptic effect on epileptiform field potentials (EFP) induced by the GABA A -antagonist bicuculline. (2) The unspecific calcium channel modulator flunarizine, which in contrast to verapamil penetrates the blood-brain barrier, depressed EFP in the low Mg 2+ -model and in the bicuculline model. (3) There was no significant difference in the antiepileptic efficacy of verapamil and flunarizine in epileptic (epilepsy surgery) and primary non-epileptic (tumor surgery) neocortical slices.

Incubation chambers for surviving brain slice preparations are in most cases designed to be stationary. For investigations on human brain tissue resected for the treatment of rain tumor or epilepsy, portable incubation chambers are needed in addition to stationary ones to allow transport of the slices between laboratories and hospitals located far from each other. For such purposes, interface chambers have been in use. In view of the ongoing discussion of the merits of interface versus submerged baths, here we describe an alternative chamber as a lightweight, easy to assemble portable bath of the submersion type for transport of surviving brain slice preparations over considerable distances. The chamber has been used in a variety of investigations on human brain slices. These slice preparations showed bioelectric properties comparable to those reported in investigations by other laboratories using stationary incubation chambers in cases where portable ones were not needed.

The action of the epileptogenic agent pentylenetetrazol (PTZ) on a cloned potassium channel of the rat brain was studied. The Kv1.1 channel was expressed in oocytes of Xenopus laevis and potassium currents were investigated in outside-out and inside-out membrane patches. The results show that PTZ increased the multi-channel potassium currents at strongly negative potentials and decreased them at potentials positive to -35 mV both in outside-out and inside-out membrane patches. The extent and manner of PTZ action, the concentration dependence as well as the onset and time course of the PTZ effect were the same both in outside-out and inside-out membrane patches. The single-channel potassium currents showed an increase in open probability and frequency of opening and a decrease in close time at -50 mV and vice versa at 0 mV with application of PTZ. The amplitude of single-channel current, the open time and the latency to the first channel opening remained almost unchanged under PTZ. The results indicate that PTZ acts via the cell membrane and influences the membrane-associated part of the potassium channel. Thereby, PTZ accelerates the transition from the inactivated to the open state of the channel at strongly negative potentials and reduces it at slightly negative and positive potentials. This mechanism may be the basis for a gate function which is in favour of the development of epileptic discharges.

The convulsive agent pentylenetetrazol (PTZ) antagonized gamma-aminobutyric acid (GABA)-induced membrane currents on RNA-injected Xenopus oocytes with both extra- and intracellular applications. With extracellular administration PTZ enters the cell within a few minutes and reaches concentrations in the millimolar range. The permeability of the plasma membrane makes it possible for systemically applied PTZ to elicit its effect on the GABA-induced currents via extra- as well as intracellular sites of action.

The effects of the epileptogenic agent pentylenetetrazol (PTZ) on current responses to glutamate (Glu) and to the Glu receptor agonist N-methyl-D-aspartate (NMDA), kainate (KA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and quisqualate (QA) were studied on RNA-injected Xenopus oocytes. PTZ antagonized the reactions to Glu as well as to NMDA, KA, AMPA and QA. With increasing concentration of AMPA, however, the depressive effect of PTZ turned to an augmentation. The complete change from a decreasing to an increasing effect of PTZ with elevated agonist concentration was unique for current responses by AMPA.

The effect of the epileptogenic agent pentylenetetrazol (PTZ) on the cloned rat brain potasssium channel Kv1.1 (labelled also RCK1) was investigated in the Xenopus laevis oocyte expression system. The Kv1.1 channel was affected by PTZ in a votlage-dependent manner. PTZ increased the potassium currents at more negative potentials and decreased them at more positive potentials. At a potential of −50 mV the potassium currents were increased by 0.97 and at −20 mV decreased by 0.21 of control value with 100 mmol/1 PTZ. The potential at which the inversion from increase to decrease occurred was −33 mV. The inactivation characteristic of the current was shifted to more negative potentials by PTZ. The PTZ effect was obtained at a threshold concentration of 1 mmol/1 and increased with rising PTZ concentrations. After removal of the tissues covering the oocyte membrane, the PTZ effect was augmented; with a concentration of 10 mmol/1 PTZ the potassium currents at 0 mV were decreased by 0.04 in oocytes with covering tissues and by 0.27 of control value in oocytes without covering tissues. Under current-clamp conditions, PTZ decreased small depolarizations and increased larger depolarizations. This effect of PTZ represents a ‘discriminatory function’ that may contribute to epileptogenesis in nervous tissues.

The aim of the present study was to test whether the organic calcium channel blocker verapamil acts not only on spontaneously occurring epileptiform field potentials (EFP) but also on EFP triggered by single electrical stimuli in the low-Mg2+ epilepsy model. The experiments were carried out on hippocampal slices of guinea pigs. EFP were elicited by omission of Mg2+ from the perfusate and recorded from stratum pyramidale and stratum radiatum in the CA1 subfield. Single electrical stimuli were applied to the Schaffer collateral pathway. Verapamil was added to the bath solution in concentrations of 40 and 60 mumol verapamil/l at normal (4 mmol/l) and elevated (8 mmol/l) K+ levels. After omission of Mg2+ from the perfusate, spontaneously occurring EFP appeared in all trials. These spontaneously occurring EFP were suppressed dose-dependently upon addition of verapamil to the perfusate. At elevated K+ levels, the latencies to suppression were significantly reduced and the dose dependency was abolished for the two doses of verapamil used. Triggered EFP reappeared upon stimulation after spontaneously occurring EFP had been suppressed, except for trials with 60 mumol verapamil/l bath solution with elevated K+ levels. The stimulus-evoked EFP were abolished with continuing perfusion of verapamil except for trials with 40 mumol/l at normal extracellular K+ concentrations. This effect was again dose dependent and enhanced by elevating the K+ level. In all experiments, stimulus-evoked EFP reappeared upon wash-out of verapamil. A primary action of verapamil on pacemaker functions in epileptogenic tissue is assumed.

The buccal ganglia ofHelix pomatia are used as model nervous structures in neurophysiological and epileptological studies. Many basic problems concerning membrane physics and the functioning of single neurons and neuronal networks can be easily studied using these ganglia. The model characecter derives mainly from the relative simplicity of this nervous system and the fact that it contains large, visually identifiable neurons. As in other invertebrate nervous systems, the large neurons have proved to be individuals showing the same functional and structural properties from one animal to another.

The diffusion of valproate (VPA) and trans -2-en-valproate were studied in agar gel and in the cerebral cortex of the rat using pressure microejection and VPA-selective microelectrodes. From the agar measurements a free diffusion coefficient for VPA of6.52×10 −6 cm 2 ·s −1 and for trans -2-en-VPA of5.25×10 −6 cm 2 ·s −1 for 37°C was determined. The tortuosity value in the cortex was 1.92 for VPA and 1.67 for trans -2-en-VPA. The tortuosity values suggest that VPA and trans -2-en-VPA diffuse mainly in the extracellular space of the brain.

In the present experiments it was tested whether the organic calcium antagonist verapamil has an influence on N- methyl- d -aspartate (NMDA) induced cortical field potentials (CFP) in neocortical slices of guinea pigs. NMDA (1 μmol/l) was applied via a micropipette by pressure pulses. Verapamil (60 μmol/l) was administered by bath application or ejected locally (100 μmol/l) and simultaneously with NMDA. Neither the systemic administration nor the local application of verapamil had an influence on NMDA induced CFP. It is concluded that the antiepileptic effect of verapamil is not mediated via the NMDA receptor.

Induction and spread of epileptic activity was studied in the buccal ganglia of Helix pomatia used as model nervous system. Epileptic activity was induced by application of epileptogenic drugs. With increasing epileptogenicity, i.e. increasing concentration of an epileptogenic drug, typical epileptic depolarizations (PDS) appeared first in the identified neuron B3 and subsequently in neurons B1 and B2. At high concentrations of the drugs large numbers of neurons generated epileptic activity. PDS were found to be primarily synchronized within the networks of electrically coupled neurons. Comparing the activities in the different networks, they were independent from one another and thus corresponded to multi-focal seizure activities in man. In the identified neuron B4 no PDS were recorded. Processes are described which are anti-epileptic and epileptogenic in effect. Thus, with enduring application of the drugs, epileptic activity decreased slowly during several hours. With repeated applications of the drugs, i.e. when there were intervals of wash-out of the drugs, epileptic activity could be increased. Comparing the activity of epileptic neurons with that of non-epileptic ones, non-epileptic neurons became progressively coupled to the epileptic rhythms. This coupling increased during up to one day. It could indicate first steps in the establishment of a generator of epileptic activity in nervous systems.

The effects of the epileptogenic substance pentylenetetrazol (PTZ) on ligand operated membrane channels were studied. For this purpose serotonin (5-HT) sensitive channels were expressed in RNA injected oocytes of Xenopus laevis . With simultaneous application of both substances, the response to 5-HT was reduced and eventually abolished by PTZ with increasing concentrations (5–100 mM). A reduction of the 5-HT response also appeared when PTZ was applied in various intervals (15 and 240 s) before 5-HT. It may be assumed that PTZ produces a component of its epileptogenic effect by acting on ligand operated membrane channels.