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Spreading depression (SD), a self-propagating depolarization of neurons and glia, is believed to play a role in different neurological disorders including migraine aura and acute brain ischaemia. Initiation and propagation of SD modulate excitability of neuronal network. A brief period of excitation heralds SD which is immediately followed first by prolonged nerve cell depression and later by an excitatory phase. The aim of the present study was to characterize local and remote transmitter receptor changes after propagation of cortical SD. Quantitative receptor autoradiography was used to asses 16 transmitter receptor types in combined striatum-hippocampus-cortex slices of the rat 1 h after induction of cortical SD. In neocortical tissues, local increases of glutamate NMDA, AMPA, and kainate receptor binding sites were observed. In addition to up-regulation of ionotropic glutamate receptors, receptor binding sites of GABA(A), muscarinic M1 and M2, adrenergic alpha(1) and alpha(2), and serotonergic 5-HT(2) receptors were increased in the hippocampus. Cortical SD also upregulated NMDA, AMPA, kainate, GABA(A), serotonergic 5-HT(2), adrenergic alpha(2) and dopaminergic D1 receptor binding sites in the striatum. These findings indicate selective changes in several receptors binding sites both in cortical and subcortical regions by SD which may explain delayed excitatory phase after SD. Mapping of receptor changes by cortical SD increases our understanding of the mechanism of SD action in associated neurological disorders.

In vitro and in vivo brain slice techniques were used to examine phencyclidine (PCP) effects on the lateral propagation of epileptiform field potentials (EFP) across adjacent areas of rat frontal neocortex. Epileptiform activity was induced by perfusing slices with Mg 2+-free artificial cerebrospinal fluid. Simultaneous field potential recordings of EFP were obtained from four microelectrodes placed 2-3 mm apart across coronal slices in the third layer. PCP, applied focally between recording sites, blocked rapid propagation across treated areas and resulted in the emergence of spatially separate, independent pacemakers. The characteristics of paroxysmal depolarization shifts did not change significantly by the blockade of lateral propagation of EFP. The same asynchronized pattern of EFP conduction was observed after local application of the N-methyl-D-aspartate (NMDA)-receptor antagonist DL-2-amino-5-phosphono-valeric acid. Local administration of haloperidol as well as NMDA before PCP application reversibly prevented appearance of multiple pacemakers. Focal application of dopamine produced an abnormal pattern of lateral conduction of EFP in 50 % of tested slices. Pacemaker failure as an indicator of functional impairment of cortical integration is the proposed mechanism for developing of schizophrenia-like psychosis associated with epilepsy. Abbreviations. APV: DL-2-amino-5-phosphono-valeric acid EEG:electroencephalogram EFP:epileptiform field potentials NMDA:N-methyl-D-aspartate PCP:phencyclidine SLPE:Schizophrenialike psychosis associated with epilepsy

Generally, increases in cortical activity go in parallel with negative shifts and decreases with positive shifts of cortical d.c. potentials. The aim of the present investigation was to test the effects of the inhibitory transmitter gamma-aminobutyric acid (GABA) and of GABA receptor agonists on cortical d.c. potentials. Concomitant changes of local pH were measured to get first insights as to the mechanisms of the evoked d.c. changes. The experiments were carried out on anesthetized and artificially ventilated rats. d.c. potentials were recorded at a cortical depth of about 1000 microm by glass microelectrodes. Extracellular pH was measured by ion-selective microelectrodes. GABA (0.1 mol/l), the GABA(A) receptor agonist muscimol (0.1 mmol/l) and the GABA(B) receptor agonist baclofen (0.1 mmol/l) were microejected by pressure pulses at a distance of 20-40 microm from the recording electrode. GABA evoked positive d.c. shifts with low pressure ejection and long application times. With increasing pressure the positive d.c. shifts were initially superimposed by negative ones. The GABA(A) receptor agonist muscimol elicited negative and the GABA(B) receptor agonist baclofen positive displacements of the d.c. potential independent of application time or pressure. The negative d.c. shifts induced by GABA and muscimol were associated with an extracellular alkalization of up to 0.1 pH units. The findings led one to assume (1) that the negative d.c. shift after GABA application was due to a neuronal depolarization and to an increase in excitation via local alkalization and (2) that the positive d.c. shift mirrored neuronal hyperpolarization.

In animal models, the hallmark of a hypoxic condition is a strong negative shift of the DC potential (anoxic terminal negativity, ATN). This DC-shift is interpreted to be primarily due to a breakdown of the membrane potential of neurons. Such massive neuronal depolarizations have not been reported for all human neocortical neurons in vitro even during prolonged hypoxic periods. This poses the question whether ATN develop also in human neocortical slices made hypoxic. ATN could be observed when human brain slice preparations (n = 15, 13 patients) were subjected to periods of hypoxia (10 to 120 min). These ATN were usually monophasic and appeared with a latency of 16 +/- 4 min (mean +/- S.E.M.). Separating the ATN according to their slopes of rise, steep (> 10 mV/min) and flat (< 10 mV/min) ATN could be distinguished. Steep and flat ATN may be regarded as two different entities of reactions since steep ATN had also greater amplitudes and slopes of decay as compared a flat ATN. With repetitive hypoxias, the latency of both the steep and flat ATN was reduced for the following hypoxic episodes. During hypoxic DC-shifts, evoked potentials were suppressed. With the 1st through 4th hypoxia, they recovered fully within 30 min after reoxygenation when hypoxia was terminated at the plateau of ATN; with extension of hypoxia, recovery was only partial. From the 5th hypoxia onwards, recovery usually did not take place or was not complete.

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 GABAA-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.

The influence of the glutamate subreceptor agonists N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) on cortical field potentials and on changes in extracellular free calcium concentration ([Ca2+]o) was tested on human neocortical slices (eleven from nine different patients). The tissue used was a small portion of that which is normally removed for the treatment of a brain tumor. [Ca2+]o and field potentials were measured by Ca(2+)-selective microelectrodes. Local pressure-microejection of NMDA (100 mumol/l)- and AMPA (1 mmol/l)-induced negative field potentials with maximal amplitudes of 0.9 +/- 0.1 mV (11 slices, mean +/- S.E.M.) and 1.0 +/- 0.1 mV (nine slices), respectively. The negative field potentials induced by NMDA were accompanied by monophasic decreases of [Ca2+]o (0.8 +/- 0.1 mmol/l, nine slices). AMPA elicited no (three slices) or only minor decreases of [Ca2+]o (0.2 +/- 0.1 mmol/l, five slices). The responses to the glutamate subreceptor agonists NMDA and AMPA were reversibly depressed by adding their specific antagonists DL-2-amino-5-phosphonovalerate (APV, 100 mumol/l, six slices) and 6-cyano-7-nitroquinoxalin-2,3-dion (CNQX, 5 mumol/l, four slices), respectively. The results correspond to findings in animal experiments and are consistent with the interpretation that in the human neocortex the Ca2+ permeability of channels gated by NMDA is higher than those gated by AMPA.

Epileptic activity induced by the GABAA receptor antagonist bicuculline is known to be blocked by organic calcium antagonists. To further analyse the mechanism underlying convulsant activity induced by substances reducing GABA-mediated synaptic transmission, the effect of organic calcium channel blockers on epileptic activity induced by the GABAA channel blocker picrotoxin in hippocampal and neocortical slices of guinea pigs were investigated. Verapamil and flunarizine suppressed paroxysmal depolarization shifts (PDS) of single neurons and accompanying epileptic field potentials (EFP). As a measure of drug action the repetition rate of epileptic events were used. The depression down to 10% the initial value (90% depression) is indicated. In the hippocampus verapamil suppressed PDS/EFP within 70 +/- 16 min (40 mumol/l) and within 39 +/- 5 min (60 mumol/l). This suppression was reversible with washout of verapamil. Flunarizine irreversibly blocked EFP/PDS within 108 +/- 14 min (18 mumol/l). In the neocortex verapamil reversibly suppressed EFP within 146 +/- 6 min (40 mumol/l) and 127 +/- 26 min (60 mumol/l). Flunarizine irreversibly blocked EFP within 181 +/- 30 min (3 mumol/l) and 109 +/- 13 min (18 mumol/l). The results suggest that voltage dependent calcium channels are essentially involved in picrotoxin-induced epileptic activity.

Free concentration of valproate (VPA) was measured simultaneously and continuously in blood and in the extracellular space of cerebral cortex of rats by VPA-selective microelectrodes. Constant amounts of VPA were injected into the femoral vein with differing duration of injection. Immediately after drug application, the concentration of free VPA in blood and brain increased to a peak value, the degree of which increased with the speed of injection. Ten to 15 min after VPA injection, a plateau value was reached. This plateau value was equal in the extracellular space of cortex and in blood. The data indicate that VPA can "freely" cross the blood-brain barrier (BBB).

Organic calcium channel blockers have been demonstrated to abolish epileptic activity in various experimental models. Furthermore, it was shown that the antiepileptic efficacy of the organic calcium channel blocker verapamil was significantly augmented when the KCl concentration background was elevated to levels normally occurring during epileptic seizures. The aim of the present investigation was to test whether flunarizine, which in contrast to verapamil is able to penetrate the blood brain barrier, suppresses epileptic activity in neocortical slice preparations, and whether this effect would be enhanced by raising the KCl background concentration. Epileptic activity was induced in neocortical slices of guinea pigs by omission of Mg2+ from the superfusate. As a measure of epileptic activity, field potentials were recorded from layers III and V. They appeared within approx 30 min after omission of Mg2+ from the bath solutions. The frequency of occurrence in normal and elevated KCl concentration was 47 +/- 10/5 min and 46 +/- 9/5 min, respectively. Flunarizine, in concentrations of 3.2 and 18 mumol/l, abolished epileptiform activity dose dependently. A 90% depression occurred within 194 +/- 27 and 376 +/- 27 min for flunarizine concentrations of 18 and 3.2 mumol/l, respectively. Elevating the KCl back-ground concentration to 8 mmol/l significantly enhanced the antiepileptic efficacy of flunarizine. Under these conditions, a 90% depression occurred within 67 +/- 14 and 165 +/- 37 min for flunarizine. Under these conditions, a 90% depression occurred within 67 +/- 14 and 165 +/- 37 min for flunarizine concentrations of 18 and 3.2 mumol/l, respectively. The experiments demonstrate that flunarizine suppresses epileptic activity in neocortical preparations, with enhanced action in elevated K+ levels.

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 of 6.52 x 10(-6) cm2.s-1 and for trans-2-en-VPA of 5.25 x 10(-6) cm2.s-1 for 37 degrees 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.

The organic calcium channel blocker verapamil has been demonstrated to block epileptic activity in various experimental models both in vitro and in vivo. The drug, however, does not pass the blood-brain barrier, so that both the oral route and intravenous administration of the drug are ruled out for antiepileptic treatment. The present investigations analyzed the effects of verapamil applied epicortically in experimental models of interictal penicillin-induced and ictal pentylenetetrazol-induced epileptic activity in rats. Such epicortical application of verapamil was ineffective in suppressing either interictal or ictal epileptic activity. To test whether this lack of effect was due to poor penetration of the substance into the cortical tissue, the diffusion characteristics of verapamil were studied in agar and in gray matter by pressure microejection and an appropriate verapamil-selective microelectrode. The diffusion could be described fully by a diffusion coefficient D (5.08 x 10(-6) cm2 x s-1), tortuosity lambda (1.51) and concentration-dependent uptake, k' (2.23 x 10(-3) s-1). Using these values, the depth-dependent concentration gradient resulting from superfusion of the substance was calculated for agar and brain. In concentration measurements done in brain tissue, however, verapamil could not be detected in cortical layers deeper than 150 microns, which did not agree with the theoretical prediction. This observation may indicate a diffusion barrier at the interface between superfusing fluid and tissue. The results indicate that epicortical administration of verapamil is not efficacious in treatment of epilepsy.

1. The antiepileptic effect of the organic calcium antagonist verapamil on low Mg2+ induced epileptiform discharges in the neocortex was tested. 2. The experiments were carried out on slices of the frontal neocortex (guinea pigs). Verapamil was tested at normal (4 mmol/l) and elevated (8 mmol/l) KCl levels. 3. Verapamil (40, 60 mumol/l) suppressed epileptiform activity in any case. 4. With elevated K+ concentration the suppressive effect of verapamil was significantly accelerated. 5. Epileptic activity reappeared when verapamil was omitted from the Mg(2+)-free superfusate.

The results demonstrate that organic calcium antagonists are able to reduce epileptic activity at the level of single neurons and of neuronal populations. This holds true also for human cortical tissue. Among other observations already published this justifies the hope that calcium antagonistic agents might be useful in the treatment of human epilepsies (28).

Long-term cultures of organotypic neonatal rat neocortex slices were maintained in a serum-free medium supplemented with 25 mM potassium. Such cultures continue to be bioelectrically silent upon return to a 5 mM potassium medium. The absence of responses to pressure ejected gamma-aminobutyric acid (GABA) and N-methyl-D-aspartate (NMDA) from neurons in treated explants suggests that one consequence of chronic depolarization is a reduction in the density of postsynaptic transmitter receptors. [3H]Muscimol binding to neocortical membrane preparations shows a large reduction in the binding of this agonist to GABAA receptors. These data show that the quantitative expression of at least one neurotransmitter receptor, the GABAA receptor, relies on voltage-dependent activity in developing neocortical neurons in vitro.

The generation of currents and potentials detectable in the space surrounding cellular elements within central nervous structures is described. The following aspects are especially taken into account: Generator structures, membrane potential changes of single neurons and of single glial cells, generation of potentials in the extracellular space, and types of cortical field potentials.

The influence of the inhibitory transmitter gamma-aminobutyric acid (GABA) on depolarizations elicited by the excitatory amino acid N-methyl-D-aspartate (NMDA) was tested in neurons of organotypic neocortical tissue cultures (newborn rat) and in CA3 neurons of the hippocampal slice (guinea pig). Drugs were applied through a 3-barrelled micropipette by pressure ejection. Applications of GABA before the ejection of NMDA increased the amplitude of the depolarizations induced by the excitatory amino acid. It is suggested that the enhancement of NMDA responses by GABA may be mainly mediated by an intracellular common pathway.

To study the relations between epileptiform potentials in the surface EEG and motor phenomena, focal interictal epileptiform discharges (FIEDs) were induced by the combined application of penicillin and penicillinase to the cortical surface of the rat. Spinal field potentials (SFPs) served as an indicator of descending activity. The following results were obtained. (1) The focus induced by the technique generated epileptiform activity within a very restricted cortical region. (2) FIEDs were accompanied by synchronized SFPs in the cervical and lumbar cord if the focus was located in the corresponding cortical motor area. With FIEDs in non-motor areas SFPs failed to occur. A unilateral focus led to SFPs on both sides of the spinal cord. (3) The transverse distribution of field potentials within the spinal cord revealed that the early negative peak of the SFP reached its maximum in the dorsal horn. (4) After the application of the drugs the development of FIEDs preceded that of SFPs by ca. 5 min. When the drug effect ceased SFPs remained often fully developed up to 10 min after the FIEDs had disappeared. (5) The temporal relationship between the cortical and spinal events showed individual and interindividual variations. (6) Anodal and cathodal electrical polarization of the cortical surface by currents sufficient to alter the shape, amplitude and polarity of the FIEDs failed to change the SFPs. The present investigations demonstrate that the cortical output from a restricted focus does not correspond to a definite epicortical field potential.

The pattern of intracortical potential distribution during focal interictal epileptiform discharges (FIED) was analysed with respect to the occurrence of descending neuronal activity to the spinal cord recorded as spinal field potentials (SFPs). The experiments were performed in rats. Epileptiform activity was elicited by application of penicillin to the motor cortex. The spread of active penicillin was limited by penicillinase in part of the experiments. (1) When penicillinase was applied 10--20 sec before penicillin to the cortical surface typical FIED appeared in the epicortical lead. During well-established focal activity they were accompanied by negative field potentials at a depth of 300 micrometers and 600 micrometers and by positive field potentials in deeper records. This pattern of intracortical potential distribution was not associated with characteristic SFPs. (2) When penicillinase was applied simultaneously with penicillin, the fully developed epicortical FIED were accompanied by negative intracortical field potentials which in this case reached a depth of 900 micrometers. In the layers below predominantly positive potential fluctuations occurred. This pattern of intracortical potential distribution was associated with characteristic SFPs. (3) Intracortical application of penicillin at a depth of 800--900 micrometers led to negative field potentials of large amplitude in all intracortical records, with the concomitant epicortical potentials being positive in polarity. In this case SFPs occurred throughout the interictal activity. Since seizure activity can be restricted to only a few cortical laminae, descending neuronal activity to the spinal cord need not be correlated with definite epicortical potentials. A prerequisite for cortical output is intracortical activity reflected negative potentials at a depth of approx. 900 micrometers.

Effects of the organic calcium antagonists verapamil and flunarizine on pentylenetetrazol induced paroxysmal depolarizations were tested in organotypic neocortical explants taken from neonatal rats. In these in vitro experiments the papaverin derivative verapamil depressed, and finally abolished, epileptic discharges in all cases. The piperazine derivative flunarizine, however, which is known to suppress epileptic discharges in hippocampal CA3 neurons (Bingmann and Speckmann 1986), showed no significant antiepileptic effects in the explanted neocortical neurons. Thus, the present findings may indicate that the suppressive action of flunarizine on the generation of paroxysmal depolarizations is restricted to distinct populations of neurons.

The serum- and glucocorticoid-dependent kinase SGK1 is regulated by alterations of cell volume, whereby cell shrinkage increases and cell swelling decreases the transcription, expression and activity of SGK1. The kinase is expressed in all human tissues studied including the brain. The present study was performed to localize the sites of SGK1 transcription in the brain, to elucidate the influence of the hydration status on SGK1 transcription and to explore the functional significance of altered SGK1 expression. Northern blot analysis of human brain showed SGK1 to be expressed in all cerebral structures examined: amygdala, caudate nucleus, corpus callosum, hippocampus, substantia nigra, subthalamic nucleus and thalamus. In situ hybridization and immunohistochemistry in the rat revealed increased expression of SGK1 in neurons of the hippocampal area CA3 after dehydration, compared with similar slices from brains of euvolaemic rats. Additionally, several oligodendrocytes, a few microglial cells, but no astrocytes, were positive for SGK1. The abundance of SGK1 mRNA in the temporal lobe, including hippocampus, was increased by dehydration and SGK1 transcription in neuroblastoma cells was stimulated by an increase of extracellular osmolarity. Co-expression studies in Xenopus laevis oocytes revealed that SGK1 markedly increased the activity of the neuronal K+ channel Kv1.3. As activation of K+ channels modifies excitation of neuronal cells, SGK1 may participate in the regulation of neuronal excitability. [ABSTRACT FROM AUTHOR]