Biomedical subjects

U Heinemann

Publications and source records attributed to U Heinemann.

At least 91 records · Page 5Linked to original sources

PubMed1999

Age-dependence of the anticonvulsant effects of the GABA uptake inhibitor tiagabine in vitro.

Epileptic syndromes frequently start at childhood and therefore it is crucial to test new anticonvulsants at immature stages of the nervous system. We compared the effects of the gamma-aminobutyric acid (GABA) uptake inhibitor tiagabine [(R)-N-(4, 4-bis(3-methyl-2-thienyl)but)3-en-1-yl nipecotic acid] on low-Mg(2+)-induced epileptic discharges in brain slices from rat pups (p 5-8) and juvenile animals (p 15-20). In tissue from rat pups, tiagabine slightly reduced epileptiform activity in hippocampal area CA1 but had no effect in the entorhinal cortex. In juvenile rats, epileptiform discharges were unaffected in CA1 but suppressed by 60% in the entorhinal cortex. While tiagabine increases its efficacy with age, in-situ hybridisation and PCR analysis show that mRNA coding for the neuronal GABA-transporter GAT-1 is already present at p 5. We therefore conclude that the increasing efficacy of tiagabine during ontogenesis is due to functional maturation of GABAergic synapses rather than to up-regulation of GAT-1 expression.

Action Potentials↗

PubMed1999

Epileptiform activity induced by 4-aminopyridine in entorhinal cortex hippocampal slices of rats with a genetically determined absence epilepsy (GAERS).

Patients with absence epilepsy frequently develop convulsions later in life. We were therefore interested whether tissue from rats with a genetic absence epilepsy is more prone to seizure generation than normal animals. We compared the epileptiform activities induced by 4-aminopyridine (4-AP) induced in hippocampal-entorhinal cortex slices from genetic absence epilepsy rats of Strasbourg (GAERS, age 6 months) in which absence seizures have been present for about 4 months and from control non epileptic rats (NE). 4-AP induced short recurrent discharges in area CA1 of rat hippocampus, seizure-like events and interictal discharges in the entorhinal cortex. The various epileptiform discharges did not differ between the two strains in amplitude, duration and frequency. However, the latency for induction of different epileptiform activities by 50 microM 4-AP was significantly shorter in GAERS (about 16 min) than in NE rats (about 25 min). We also analysed differences in evoked field potentials (fp) in hippocampal area CA1 before, during and after application of 4-AP. Before application of 4-AP, responses to stimulation of Schaffer collateral were smaller in GAERS than in NE rats. Paired pulse potentiation was significantly larger in GAERS than in NE rats. 4-AP in the bath augmented the size of the evoked field potentials and this increase was larger in GAERS than in NE rats. Our findings show a greater excitability of hippocampal area CA1 in GAERS rats and a greater ability to develop 4-AP-induced epileptiform activity in combined hippocampal-enthorhinal cortex slices in GAERS than in NE rats.

4-Aminopyridine↗

PubMed1999

Effects of ionotropic glutamate receptor blockade and 5-HT1A receptor activation on spreading depression in rat neocortical slices.

The effect of the AMPA antagonist NBQX (10 microM), NMDA antagonist ketamine (100 microM) and 5-HT1A agonist 8-OH-DPAT (1, 10 and 100 microM) on the properties of a KCl-induced spreading depression (SD) was studied in parietal cortical slices of adult rats. Whereas NBQX did not significantly affect the SD, ketamine significantly (p < 0.01) reduced the amplitude of the first SD peak (12.8 +/- 4.6 mV) and blocked the second SD peak when compared with the controls (19.8 +/- 5.2 mV and 25 +/- 5 mV, respectively). Ketamine also decreased the SD duration at half maximal amplitude from 34.9 +/- 12.4 s to 22.2 +/- 12 s (p < 0.05). 8-OH-DPAT attenuated the duration of the SD from 42 +/- 15.6 s to 21.2 +/- 10.6 s (p < 0.05, 100 microM). These data indicate that not only NMDA receptor blockade, but also activation of the 5-HT1A receptor attenuates the SD and may be beneficial in the reduction of ischemic injury following focal cerebral ischemia.

Animals↗

PubMed1999

Anoxic decrease in potassium outward currents of hippocampal cultured neurons in absence and presence of dithionite.

The effect of brief anoxia on voltage dependent K(+)-currents of hippocampal cultured neurons was studied. The oxygen scavenger dithionite (hydrosulphite) was previously used for creating zero oxygen pressure. However, dithionite consumes O(2) in parallel with generation of superoxide radicals and is a strongly reducing agent. In this study anoxia was produced by perfusion of the neurons with a solution bubbled with nitrogen for 1 h using a chamber with an argon layer isolating the anoxic bath flow from atmospheric oxygen in presence and absence of dithionite. Oxygen partial pressure of dithionite-free solution was determined by oxygen dependent quenching of the phosphorescence of Pd-coproporphyrin to be 0.15+/-0. 02 Torr (values are given as mean+/-S.D., n=6). Slow (I(K))- and fast (I(A))-inactivating K(+)-currents were measured with the patch clamp technique in the whole cell configuration. Exposure of the neurons to anoxia reversibly decreased the amplitude of I(K) at a test pulse of 0 mV to 77+/-12% (n=7) in absence and to 83+/-7% (n=6) in presence of 2 mM dithionite; the amplitude of I(A) decreased to 78+/-11% in absence and to 82+/-9% in presence of 2 mM dithionite. Voltage dependence of activation and inactivation shifted 5 min after exposure to anoxia reversibly by about 6 mV in depolarizing direction. The decay times of inactivation were insensitive to anoxia. Dithionite had no significant effects on K(+)-currents. In 15 of 21 neurons not employed for analysis on K(+)-currents, a reversible increase in holding current under dithionite was observed. In absence of dithionite in 4 of 19 neurons the holding current reversibly increased during anoxia. Although dithionite does not affect K(+)-currents, changes in holding current show that the dithionite may affect neurons independently of oxygen deprivation.

Animals↗

PubMed1999

Epileptiform discharges induced by combined application of bicuculline and 4-aminopyridine are resistant to standard anticonvulsants in slices of rats.

Application of 4-aminopyridine (4AP) has previously been reported to produce different patterns of epileptiform discharges in entorhinal cortex-hippocampal-slices. Here we describe that 4-AP induced epileptiform activity in the EC becomes insensitive to anticonvulsant drugs (phenytoin, carbamazepine, valproic acid, phenobarbital) when GABAergic transmission is blocked by bicuculline. We propose that the activities induced by 4-aminopyridine and bicuculline may provide an in vitro model for the development of new drugs against difficult-to-treat focal epilepsy.

4-Aminopyridine↗

PubMed1999

Acute cell damage after low Mg2+-induced epileptiform activity in organotypic hippocampal slice cultures.

Upon perfusion with Mg2+-free artificial cerebrospinal fluid (ACSF) organotypic hippocampal slice cultures develop seizure-like events and tonic recurrent discharges in which areas CA3 and CA1 and, in contrast to acute slices, also the dentate gyrus (DG) participate. Using the fluorescent dye propidium iodide (PI) we show that sustained epileptic activity causes cell death in the DG and pyramidal cell layer particularly evident in the granule cell layer of the DG. This correlates with the decrease of the electrophysiological responses to hilar stimulation. Interestingly, perfusion with carbogenated serum-free ACSF also induces some cell death which is, however, mild compared with low magnesium treated slice cultures.

Animals↗

PubMed1999

Synaptic reorganization in explanted cultures of rat hippocampus.

Due to loss of afferent innervation, synaptic reorganization occurs in organotypic hippocampal slice cultures. With extra- and intracellular recordings, we confirm that the excitatory loop from the dentate gyrus (DG) to CA3 and further to CA1 is preserved. However, hilar stimulation evoked antidromic population spikes in the DG which were followed by a population postsynaptic potential (PPSP); intracellularly, an antidromic spike with a broad shoulder or EPSP/IPSP sequences were induced. Synaptic responses were blocked by glutamate receptor antagonists. Stimulation of CA1 induced a PPSP in DG. Dextranamine stained pyramidal cells of CA1 were shown to project to DG. After removal of area CA3, DG's and mossy fibers' (MF) stimulation still elicited PPSPs and EPSP/IPSP sequences in area CA1 which disappeared when a cut was made through the hippocampal fissure. During bicuculline perfusion, hilar stimulation caused EPSPs in granule cells and spontaneous and evoked repetitive firing appeared even after its isolation from areas CA3 and CA1. Collateral excitatory synaptic coupling between granule cells was confirmed by paired recordings. Besides the preservation of the trisynaptic pathway in this preparation, new functional synaptic contacts appear, presumably due to MF collateral sprouting and formation of pathways between areas CA1 and DG.

2-Amino-5-phosphonovalerate↗

PubMed1999

Epileptiform activity induced by low Mg2+ in cultured rat hippocampal slices.

Organotypic cultured slices of the rat hippocampus undergo synaptic reorganization. Besides the establishment of reciprocal connections between area CA1 and the dentate gyrus (DG), collateral excitatory connections between granule cells are formed which are similar to those appearing in several epilepsy models and in the DG from patients with temporal lobe epilepsy. We studied the characteristics of epileptiform activity induced by low Mg2+ perfusion in cultured hippocampal slices using extra- and intracellular recordings. With low Mg2+ perfusion synchronous seizure like events (SLEs) were readily observed in the DG and areas CA3 and CA1. Also, the isolated DG was able to display seizure like activity. Intracellular recordings revealed long lasting depolarization shifts in granule cells of the DG and pyramidal cells of areas CA3 and CA1. The SLEs, lasting 2-3 s, could be recorded for at least 3 h in areas CA1 and CA3. However, approximately an hour after perfusion with low Mg2+, the epileptiform activity disappeared in the DG and responses to single pulse hilar stimulation progressively deteriorated. These responses returned to control values 1 week after reincubating the cultures. Interestingly, no deterioration of stimulus induced responses was observed in the isolated DG after exposure to low Mg2+.

Animals↗

PubMed1999

Effects of glutamate uptake blockers on stimulus-induced field potentials in rat entorhinal cortex in vitro.

L-Glutamic acid (Glu) is a key excitatory transmitter in the central nervous system. Excessive amounts of Glu are highly toxic to neurons and particularly the entorhinal cortex (EC) exhibits a remarkable loss of cells in the superficial layers in acute brain injury. The accumulation of Glu is limited by a family of high-affinity Glu transporters. Using extracellular potential recordings in rat brain slices we tested whether application of the Glu uptake blockers dihydrokainate and L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-2,4-PDC) affect stimulus-induced field potentials (FPs) in superficial layer III and deep layer V of the medial EC. We found that a high concentration (400 microM) of the uptake blockers significantly reduces stimulus-induced FPs in both layers. At lower concentration (200 microM), only dihydrokainate is efficient. The data show that Glu uptake is involved in the control of extracellular Glu levels during synaptic excitation of layers III and V of the medial EC.

Amino Acid Transport System X-AG↗

PubMed1999

Intrinsic theta-frequency membrane potential oscillations in layer III/V perirhinal cortex neurons of the rat.

The firing of a proportion of neurons in the in vivo perirhinal cortex, a brain region involved in object recognition memory, has recently been shown to be synchronized with hippocampal theta activity. The purpose of the present study was to determine whether neurons located in perirhinal cortex have intrinsic properties that might encourage their participation in theta activity. To these ends, current clamp recordings were made from 98 neurons located in layer III/V of the in vitro rat perirhinal cortex. The intrinsic properties of these neurons were investigated, and a subset of 61 neurons were tested for the presence of membrane potential oscillations at threshold levels of depolarization. Thirty-nine percent of these neurons displayed a theta-frequency membrane potential oscillation (MPO; mean frequency = 8.6 Hz). When depolarized past spike threshold, these neurons tended to fire in clusters, with a within-cluster interspike interval close to the peak to peak interval of the MPOs. Neurons that did not generate MPOs generated nonaccomodating action potential trains with a frequency that spanned the theta range. Biocytin staining indicated that MPOs could be generated in cells with both pyramidal and nonpyramidal morphology. These findings demonstrate that a large proportion of perirhinal neurons exhibit intrinsic properties that could assist in the entrainment and synchronization of theta-frequency oscillations. These properties may enhance the communication of information between the perirhinal cortex, entorhinal cortex, and hippocampus.

Animals↗

PubMed1999

Effects of retigabine (D-23129) on different patterns of epileptiform activity induced by 4-aminopyridine in rat entorhinal cortex hippocampal slices.

The purpose of this study was to evaluate the effects of the new anticonvulsant drug N-(2-amino-4-[fluorobenzylaminol-phenyl) carbamic acid ethyl ester (retigabine, D-23129, ASTA Medica, Dresden, Germany) on different patterns of epileptiform activity induced by 4-aminopyridine (4AP) in rat entorhinal cortex hippocampal slices. Application of 4AP (100 microM) induced in entorhinal cortex two different types of epileptiform activities; seizure-like events (SLE) and interictal epileptiform discharges (IED). Bicuculline (10 microM) changed 4AP-induced SLE and IED to recurrent epileptiform discharges (RED). IED were isolated after blockade of the SLE by glutamate receptor antagonists for alpha-amino-3-hydroxy-5-methylisoxazole4-proprionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors, i.e. 1,2,3,4 tetrahydro-6-nitro-2,3-dioxo-benzolflquinoxaline-7-sulfonamide (NBQX, 10 microM) and 2-amino-5-phosphonovaleric acid (APV, 30 microM). Anticonvulsant properties of retigabine were evaluated as effect on the frequency and amplitude of SLE, IED and RED. Retigabine suppressed all types of epileptiform events in a dose dependent and reversible manner. SLE were suppressed in 71.4 and 100% of slices by 5 and 10 microM, respectively. The frequency of IED was significantly reduced by 20 microM retigabine (40.9+/-24.5%) and IED were blocked completely by 50 microM retigabine. When IED were isolated by application of glutamate antagonists 20 microM retigabine was sufficient to block this activity completely. RED induced by combined application of bicuculline and 4AP were blocked in 71.4% of the tested slices with 100 microM retigabine. The frequency of the RED in the remaining slices was reduced by 96.1+/-6.1%. We conclude that retigabine acts on a large variety of different epileptiform activities in temporal lobe structures that are known to develop readily pharmacoresistant seizures.

4-Aminopyridine↗

PubMed1999

Two electrophysiologically distinct types of granule cells in epileptic human hippocampus.

We investigated the electrophysiology of morphologically identified human granule cells with conventional current-clamp recordings. Slices were prepared from 14 human epileptic sclerotic hippocampi. Granule cells appeared to have a diverse electrophysiology. Each cell was distinguished by the shape of the afterhyperpolarization following single action potentials. Two types could be discerned: type I afterhyperpolarizations were monophasic and brief (typically 10-40 ms), whilst type II afterhyperpolarizations were biphasic and long (typically 50-100 ms). The two types also differed in their repetitive firing behaviour and action potential morphology: type I cells had significantly weaker spike frequency adaptation, lower action potential amplitude and smaller action potential upstroke/downstroke ratio. Thus, the firing pattern of type I cells resembled that of rodent dentate interneurons. In contrast, the corresponding parameters of type II cells were comparable to rodent dentate granule cells. Despite the distinct firing patterns, membrane properties were not different. The two types of cells also differed in their synaptic responses to stimulation of the perforant path. At strong suprathreshold stimulation intensity, type I cells always generated multiple action potentials, whereas type II cells usually spiked once only. Slow inhibitory postsynaptic potentials were not detected in type I neurons, but were easily identified in type II neurons. Extracellular recordings of perforant path-evoked field potentials in the cell layer confirmed that the majority of granule cells showed multiple discharges even when we recorded simultaneously from a type II cell that generated one action potential only. The morphology of both types of cells was characteristic of what has been described for primate dentate granule cells. Based on comparisons with previous studies on rodent and human granule cells, we tentatively hypothesize that: (i) the majority of granule cells from sclerotic hippocampus display an hyperexcitable epileptogenic electrophysiology; (ii) there is a subset of granule cells whose electrophysiology is preserved and is more comparable to granule cells from non-epileptic hippocampus.

Adult↗

PubMed1999

Anticonvulsant actions of furosemide in vitro.

Anticonvulsant properties of furosemide have been suggested to reduce neuronal synchronization via its inhibitory effect on the Na+/K+/2Cl- co-transport system. We have studied effects of furosemide on spontaneous epileptiform activity and analysed effects of furosemide on amplitudes of stimulus-induced population-spikes, on stimulus-induced K+ changes, on extracellular pH changes at rest and during stimulation, and on changes in the extracellular space-volume. We used three different in vitro models of epilepsy in the combined hippocampal-entorhinal cortex slice preparation. Furosemide reversibly suppressed low Ca2+-induced epileptiform activity in hippocampus proper and blocked or significantly reduced different types of epileptiform discharges in the low Mg2+ model and the 4-aminopyridine model. Amplitudes of evoked field potentials underwent an initial slight increase followed by a significant reduction after prolonged treatment with furosemide. Stimulus-induced increases in extracellular potassium were also significantly reduced. Furosemide caused an alkaline shift at rest. Stimulus-induced pH transients changed from a biphasic alkalotic-acidotic sequence to a monophasic alkalotic shift. Stimulation-induced shrinkage of extracellular space-volume was reduced by furosemide, whereas no effect on baseline extracellular space-volume was seen. We conclude, that furosemide possesses strong anticonvulsive effects in various in vitro models of epilepsy. The anticonvulsive properties of furosemide cannot be explained by its effects on extracellular pH changes but appear in part to be mediated via a reduced excitability with consequent reduction of activity-induced potassium rises. Finally, partial inhibition of activity-induced extracellular space shrinkage may contribute to its anticonvulsant properties.

4-Aminopyridine↗

PubMed1999

Voltage-gated proton currents in microglia of distinct morphology and functional state.

Whole-cell patch-clamp measurements were performed to investigate voltage-gated proton currents (I(PR)) in cultured murine microglia of distinct morphology and functional state. We studied I(PR) in ameboid microglia of untreated cultures, in ameboid microglia which had been activated by lipopolysaccharide, and in ramified microglia which had been exposed to astrocyte-conditioned medium. Proton currents of these three microglia populations did not differ regarding their activation threshold or the voltage dependence of steady-state activation. Moreover, pharmacological properties of I(PR) were similar: proton currents were sensitive to extracellularly applied Zn2+ or La3+, and could be abolished by each of those at a concentration of 100 microM. In the presence of extracellular Na+, I(PR) was decreased to a similar small extent due to activity of the Na+/H+ exchanger in all microglial populations. In contrast, proton currents of microglia differed between the three cell populations with respect to their current density and their time-course of activation: in comparison with untreated microglia, the current density of I(PR) was reduced by about 50% in microglia after their treatment with either lipopolysaccharide or astrocyte-conditioned medium. Moreover, I(PR) activated significantly more slowly in cells exposed to lipopolysaccharide or astrocyte-conditioned medium than in untreated cells. It can be concluded that the distinct H+ current characteristics of the three microglial populations do not correlate with the functional state of the cells.

Animals↗

PubMed1999

Excitatory actions of NMDA receptor antagonists in rat entorhinal cortex and cultured entorhinal cortical neurons.

We have characterized excitatory effects of non-competitive NMDA receptor antagonists MK-801, PCP, and ketamine in the rat entorhinal cortex and in cultured primary entorhinal cortical neurons using expression of immediate early gene c-fos as an indicator. NMDA receptor antagonists produced a strong and dose-dependent increase in c-fos mRNA and protein expression confined to neurons in the layer III of the caudal entorhinal cortex. Induction of c-fos mRNA is delayed and it is inhibited by antipsychotic drugs. Cultured entorhinal neurons are killed by high doses of MK-801 and PCP but c-fos expression is not induced in these neurons indicating that this in vitro model does not fully replicate the in vivo effects of PCP-like drugs in the entorhinal cortex. Excitatory effects of the NMDA receptor antagonists may be connected with the psychotropic side effects of these drugs and might become a useful model system to investigate neurobiology of psychosis.

Animals↗

PubMed1999

Crystal structure of acceptor stem of tRNA(Ala) from Escherichia coli shows unique G.U wobble base pair at 1.16 A resolution.

The acceptor stem of Escherichia coli tRNA(Ala), rGGGGCUA.rUAGCUCC (ALAwt), contains the main identity element for the correct aminoacylation by the alanyl tRNA synthetase. The presence of a G3.U70 wobble base pair is essential for the specificity of this reaction, but there is a debate whether direct minor-groove contact with the 2-amino group of G3 or a distortion of the acceptor stem induced by the wobble pair is the critical feature recognized by the synthetase. We here report the structure analysis of ALAwt at near-atomic resolution using twinned crystals. The crystal lattice is stabilized by a novel strontium binding motif between two cis-diolic O3'-terminal riboses. The two independent molecules in the asymmetric unit of the crystal show overall A-RNA geometry. A comparison with the crystal structure of the G3-C70 mutant of the acceptor stem (ALA(C70)) determined at 1.4 A exhibits a modulation in ALAwt of helical twist and slide due to the wobble base pair, but no recognizable distortion of the helix fragment distant from the wobble base pair. We suggest that a highly conserved hydration pattern in both grooves around the G3.U70 wobble base pair may be functionally significant.

Base Pairing↗

PubMed1999

Potent depression of stimulus evoked field potential responses in the medial entorhinal cortex by serotonin.

1. The entorhinal cortex (EC), main input structure to the hippocampus, gets innervated by serotonergic terminals from the raphe nuclei and expresses 5-HT-receptors at high density. Using extra- and intracellular recording techniques we here investigated the effects of serotonin on population and cellular responses within the EC. 2. Stimulation in the lateral entorhinal cortex resulted in complex field potential responses in the superficial EC. The potentials are composed of an early antidromic and a late orthodromic component reflecting the efferent and afferent circuitry. 3. Serotonin (5-HT) reduced synaptic potentials of the stimulus evoked extracellular field potential at all concentrations tested (0. 1 - 100 microM; 59%-depression by 10 microM serotonin), while the antidromic response was not significantly changed by up to 50 microM 5-HT. Depression of field potential responses by serotonin was associated with a significant increase in paired-pulse facilitation from 1.15 to 1.88. 4. The effects of serotonin on field potential responses were mimicked by 5-HT1A-receptor agonists (8-OH-DPAT, 5-CT) and partially prevented by the 5-HT1A-receptor antagonist (S-UH-301). Moreover, the 5-HT1A-receptor antagonist WAY100635 reduced the effect of 5-CT. 5. Fenfluramine, a serotonin releaser, mimics the effects of serotonin on stimulus-evoked field potential responses, indicating that synaptically released serotonin can produce the changes in reactivity to afferent stimulation. 6. Depression of isolated AMPA-receptor mediated EPSCs by serotonin as well as fenfluramine was associated with an increase in paired pulse facilitation, indicating a presynaptic locus of action. 7. We conclude that physiological concentrations of serotonin potently suppresses excitatory synaptic transmission in the superficial entorhinal cortex by a presynaptic mechanism.

Animals↗

PubMed1999

Intrinsic optical signal measurements reveal characteristic features during different forms of spontaneous neuronal hyperactivity associated with ECS shrinkage in vitro.

We induced three different forms of spontaneous synchronous hyperactivity in adult rat hippocampal-entorhinal cortex slices in order to investigate effects on the intrinsic optical signal and associated changes in the extracellular space (ECS) volume. Low-Mg2+ artificial cerebrospinal fluid (ACSF) and the addition of 4-aminopyridine induced synchronous hyperactivity resulting mainly from increased synaptic transmission, while low-Ca2+ ACSF induced hyperactivity in the absence of evoked synaptic transmission. In the two models of enhanced synaptic transmission, spontaneous activity lead to an immediate increase of light transmission. In contrast, a decrease of light transmission took place during low-Ca2+-induced hyperactivity. All three forms of synchronous neuronal hyperactivity were associated with a shrinkage of the ECS volume, as revealed by the tetraethylammonium signal, measured with ion-sensitive microelectrodes. This indicates that the change in the intrinsic optical signal is not simply related to a shrinkage in ECS volume. We conclude that different forms of spontaneous synchronous neuronal hyperactivity are associated with characteristic optical signals and that the direction of the change in intrinsic optical signal does not reflect ECS shrinkage alone.

4-Aminopyridine↗