Biomedical subjects

Uwe Heinemann

Publications and source records attributed to Uwe Heinemann.

At least 55 records · Page 3Linked to original sources

PubMed2003

Metabotropic receptor-mediated Ca2+ signaling elevates mitochondrial Ca2+ and stimulates oxidative metabolism in hippocampal slice cultures.

Metabotropic receptors modulate numerous cellular processes by intracellular Ca2+ signaling, but less is known about their role in regulating mitochondrial metabolic function within the CNS. In this study, we demonstrate in area CA3 of rat organotypic hippocampal slice cultures that glutamatergic, serotonergic, and muscarinic metabotropic receptor ligands, namely trans-azetidine-2,4-dicarboxylic acid, alpha-methyl-5-hydroxytryptamine, and carbachol, transiently increase mitochondrial Ca2+ concentration ([Ca2+]m) as recorded by changes in Rhod-2 fluorescence, stimulate mitochondrial oxidative metabolism as revealed by elevations in NAD(P)H fluorescence, and induce K+ outward currents as monitored by rapid increases in extracellular K+ concentration ([K+]o). Carbachol (1-1,000 microM) elevated NAD(P)H fluorescence by <or=14%DeltaF/F0 and increased [K+]o by <or=4.3 mM in a dose-dependent manner. Carbachol-induced responses persisted in Ca2+-free solution and blockade of ionotropic glutamatergic and nicotinic receptors. Under similar conditions caffeine, known to cause Ca2+-induced Ca2+ release (CICR), also evoked elevations in [Ca2+]m, NAD(P)H fluorescence and [K+]o that, in contrast to carbachol-induced responses, displayed oscillations. After depletion of intracellular Ca2+ stores by carbachol in Ca2+-free solution, re-application of 1.6 mM Ca2+-containing solution triggered marked elevations in [Ca2+]m, NAD(P)H fluorescence and [K+]o. These data indicate that metabotropic transmission effectively regulates mitochondrial oxidative metabolism via diverse receptor types in hippocampal cells and that inonitol 1,4,5-trisphosphate-induced Ca2+ release (IICR) or CICR or capacitative Ca2+ entry might suffice in stimulating oxidative metabolism by elevating [Ca2+]m. Thus activation of metabotropic receptors might significantly contribute to generation of ATP within neurons and glial cells.

Animals↗

PubMed2003

Accelerated hippocampal spreading depression and enhanced locomotory activity in mice with astrocyte-directed inactivation of connexin43.

Using a human glial fibrillary acidic protein (hGFAP) promoter-driven cre transgene, we have achieved efficient inactivation of a floxed connexin43 (Cx43) gene in astrocytes of adult mice. The loss of Cx43 expression was monitored in a cell-autonomous manner via conditional replacement of the Cx43-coding region by a lacZ reporter gene. In this way, we bypassed the early postnatal lethality previously reported for Cx43 null mice and characterized the phenotypic consequences of Cx43 deficiency in the CNS. Mice lacking Cx43 in astrocytes were viable and showed no evidence of either neurodegeneration or astrogliosis. Spreading depression (SD) is a pathophysiological phenomenon observed in the CNS that is characterized by a propagating wave of depolarization followed by neuronal inactivation. Inhibitors of gap junctional communication have previously been shown to block initiation and propagation of SD. In contrast, we observed an increase in the velocity of hippocampal SD in the stratum radiatum of mice lacking Cx43 in astrocytes. In the same brain subregion, dye-coupling experiments revealed a reduction in overall astrocytic intercellular communication by approximately 50%. This strongly suggests separate and different neuronal and glial contributions of gap junctional intercellular communication to SD. Concomitant with increased velocity of spreading depression, we observed enhanced locomotory activity in mice lacking Cx43 in astrocytes.

Animals↗

PubMed2003

A novel mechanism underlying drug resistance in chronic epilepsy.

The development of resistance to pharmacological treatment is common to many human diseases. In chronic epilepsy, many patients develop resistance to anticonvulsant drug treatment during the course of their disease, with the underlying mechanisms remaining unclear. We have studied cellular mechanisms underlying drug resistance in resected hippocampal tissue from patients with temporal lobe epilepsy by comparing two groups of patients, the first displaying a clinical response to the anticonvulsant carbamazepine and a second group with therapy-resistant seizures. Using patch-clamp recordings, we show that the mechanism of action of carbamazepine, use-dependent block of voltage-dependent Na(+) channels, is completely lost in carbamazepine-resistant patients. Likewise, seizure activity elicited in human hippocampal slices is insensitive to carbamazepine. In marked contrast, carbamazepine-induced use-dependent block of Na(+) channels and blocked seizure activity in vitro in patients clinically responsive to this drug. Consistent with these results in human patients, we also show that use-dependent block of Na(+) channels by carbamazepine is absent in chronic experimental epilepsy. Taken together, these data suggest that a loss of Na(+) channel drug sensitivity may constitute a novel mechanism underlying the development of drug-resistant epilepsy.

Adult↗

PubMed2003

Kindling alters entorhinal cortex-hippocampal interaction by increased efficacy of presynaptic GABA(B) autoreceptors in layer III of the entorhinal cortex.

We studied the effect of kindling, a model of temporal lobe epilepsy, on the frequency-dependent information transfer from the entorhinal cortex to the hippocampus in vitro. In control rats repetitive synaptic activation of layer III projection cells resulted in a frequency dependent depression of the synaptic transfer of action potentials to the hippocampus. One-to-two-days after kindling this effect was strongly reduced. Although no substantial change in synaptic inhibition upon single electrical stimulation was detected in kindled rats, there was a significant depression in the prolonged inhibition following high frequency stimulation. In kindled animals, paired-pulse depression (PPD) of stimulus-evoked IPSCs in layer III neurons was significantly stronger than in control rats. The increase of PPD is most likely caused by an increased presynaptic GABA(B) receptor-mediated autoinhibition. In kindled animals activation of presynaptic GABA(B) receptors by baclofen (10 microM) suppressed monosynaptic IPSCs significantly more than in control rats. In contrast, activation of postsynaptic GABA(B) receptors by baclofen was accompanied by comparable changes of the membrane conductance in both animal groups. Thus, in kindled animals activation of the layer III-CA1 pathway is facilitated by an increased GABA(B) receptor-mediated autoinhibition leading to an enhanced activation of the monosynaptic EC-CA1 pathway.

Action Potentials↗

PubMed2003

Dopamine depresses cholinergic oscillatory network activity in rat hippocampus.

The dopaminergic neuronal system is implicated in cognitive processes in a variety of brain regions including the mesolimbic system. We have investigated whether dopamine also affects synchronized network activity in the hippocampus, which has been ascribed to play a pivotal role in memory formation. Gamma frequency (20-80 Hz) oscillations were induced by the cholinergic agonist carbachol. Oscillatory activity was examined in area CA3 of Wistar rat hippocampal slices, employing field potential and intracellular recordings. Application of carbachol initiated synchronized population activity in the gamma band at 40 Hz. Induced gamma activity persisted over hours and required GABAA receptors. Dopamine reversibly decreased the integrated gamma band power of the carbachol rhythm by 62%, while its frequency was not changed. By contrast, individual pyramidal cells recorded during carbachol-induced field gamma activity exhibited theta frequency (5-15 Hz) membrane potential oscillations that were not altered by dopamine. The dopamine effect on the field gamma activity was mimicked by the D1 receptor agonist SKF-383393 and partially antagonized by the D1 antagonist SCH-23390. Conversely, the D2 receptor agonist quinpirole failed to depress the oscillations, and the D2 antagonist sulpiride did not prevent the suppressive dopamine effect. The data indicate that dopamine strongly depresses cholinergic gamma oscillations in area CA3 of rat hippocampus by activation of D1-like dopamine receptors and that this effect is most likely mediated via impairment of interneurons involved in generation and maintenance of the carbachol-induced network rhythm.

Acetylcholine↗

PubMed2003

Ca2+-independent muscarinic excitation of rat medial entorhinal cortex layer V neurons.

Cholinergic activation of entorhinal cortex (EC) layer V neurons plays a crucial role in the medial temporal lobe memory system and in the pathophysiology of temporal lobe epilepsy. Here, we demonstrate that muscarinic activation by focal application of carbachol depolarizes EC layer V neurons and induces epileptiform activity in rat brain slices. These seizure-like bursts are associated with a somatic [Ca2+]i increase of 293 +/- 82 nm and are blocked by the glutamate receptor antagonists CNQX and APV. Muscarinic activation did not directly evoke a [Ca2+]i increase, but subthreshold and suprathreshold depolarization did. Functional axon mapping revealed local axon branching as well as axon collaterals ascending to layers II and III. During blockade of ionotropic glutamatergic AMPA and NMDA receptors, carbachol depolarized layer V neurons by +7.5 +/- 3.4 mV. This direct muscarinic depolarization was associated with a conductance increase of 35 +/- 10.3% (+4.3 +/- 1.25 nS). Intracellular buffering of [Ca2+]i changes did not block this depolarization, but prolonged action potential duration and reduced adaptation of action potential firing. The muscarinic depolarization was neither blocked by combining intracellular Ca2+-buffering (EGTA or BAPTA) with non-specific Ca2+-channel inhibition by Ni+ (1 mm), nor by Ba2+ (1 mm) nor during inhibition of the h-current by 2 mm Cs+. In whole-cell patch-clamp recording, reversal of the muscarinic current occurred at about -45 mV and -5 mV with complete substitution of intrapipette K+ with Cs+. Thus, muscarinic depolarization of EC layer V neurons appears to be primarily mediated by Ca2+-independent activation of non-specific cation channels that conduct K+ about three times as well as Na+.

Acetylcholine↗

PubMed2002

Erythropoietin improves synaptic transmission during and following ischemia in rat hippocampal slice cultures.

Erythropoietin (EPO) prevents neuronal damage following ischemic, metabolic, and excitotoxic stress. In this study evoked extracellular field potentials (FP) were used to investigate the effect of EPO on synaptic transmission in hippocampal slice cultures. EPO treated cultured slices (40 units/ml for 48 h) showed significantly increased FP during and following oxygen and glucose deprivation compared with untreated control slices. The addition of the Jak2 inhibitor AG490 (50 microM for 48 h) blocked the EPO effect. These data suggest that EPO improves synaptic transmission during and following ischemia in hippocampal slice cultures.

Animals↗

PubMed2002

Far field effects of seizure like events induced by application of 4-AP in combined entorhinal cortex hippocampal slices.

Epileptiform activity induced by 4-AP in hippocampal area CA1 is characterised by short recurrent discharges. These are occasionally superimposed by slow field potential (fp) shifts. Simultaneous recordings of fps and [K(+)](o) in area CA1 and temporal cortex showed a slow fp shift in both regions, but associated rises in [K(+)](o) occurred only in the cortex. Slow fps in area CA1 persisted after disruption of the perforant path, but were abolished after removal of the adjacent cortex from the hippocampus. These findings suggest that slow fps in CA1 can represent far field effects of seizure like events generated in neighbouring cortex.

4-Aminopyridine↗

PubMed2002

Acute neuronal injury after hypoxia is influenced by the reoxygenation mode in juvenile hippocampal slice cultures.

In neonates asphyxia is usually followed by hyperoxia due to resuscitation procedures. In order to study whether hyperoxic reoxygenation might cause additional cell injury we subjected organotypic hippocampal slice cultures of juvenile rats to normoxic or hyperoxic reoxygenation (19 or 85% oxygen, respectively) following hypoxia (3% oxygen) for 30, 60, and 120 min. Cell injury was quantified by lactate dehydrogenase (LDH) release and propidium iodide (PI) fluorescence 1 h after end of the reoxygenation period. In both experimental groups, LDH activity was significantly enhanced by hypoxia as compared to normoxic controls. However, hyperoxic reoxygenation caused a larger increase in LDH activity than normoxic reoxygenation (e.g., by a factor of 1.60 vs. 1.29 following 120 min hypoxia). PI fluorescence increased after hypoxia in all principal cell layers of the hippocampus but again showed a larger enhancement after hyperoxic reoxygenation as compared to normoxic reoxygenation (e.g., by a factor of 3.9 vs. 1.7 for CA1 and 120 min of hypoxia). After normoxic reoxygenation, PI fluorescence intensity was lower in the dentate gyrus as compared to CA1 and CA3, while it reached similar values like CA1 after high oxygen supply. In conclusion, juvenile hippocampal slice cultures subjected to hyperoxic reoxygenation display a greater amount of acute neuronal injury than slice cultures undergoing normoxic reoxygenation.

Age Factors↗

PubMed2002

Calcium currents in rat entorhinal cortex layer II stellate and layer III pyramidal neurons in acute brain slice.

The entorhinal cortex (EC) is an essential relay station in neocortical - hippocampal information transfer and memory functions. Layer II stellate and layer III pyramidal neurons show specific damage in Alzheimer's disease and epilepsy, respectively. Using whole cell patch clamp recording in rat brain slices we here demonstrate that high voltage activated Ca(2+)-currents (I(Ca)(2+)) are about 1.6-fold bigger in stellate cells than in pyramidal neurons while current density is equal in both cell types. In stellate cells I(Ca)(2+) shows stronger inactivation with depolarization, block of I(Ca)(2+) by Ni(2+) (300 and 600 microM) is more effective, and this block decreases more for currents evoked from a less negative holding potential than in layer III pyramidal neurons. These data indicate distinct molecular composition of Ca(2+)-channels and can partially explain stronger increases of [Ca(2+)](i) during 10 Hz firing activity in EC pyramidal versus stellate neurons.

Animals↗

PubMed2002

Glutamate-induced gamma oscillations in the dentate gyrus of rat hippocampal slices.

In this paper we show that gamma oscillations can be elicited by brief (< or = 200 ms) local applications of glutamate in the dentate gyrus of rat hippocampal slices. Dentate gamma oscillations show an initial peak frequency of approximately 70 Hz and last for up to 4 min. The network activity involves functional GABA(A) receptors as it is drastically reduced by GABA(A) receptor antagonists. The oscillations can be observed in the whole dentate gyrus-CA3-network and are coherent between the dentate gyrus and area CA3 for variable periods. Thus, long-lasting gamma oscillations can be experimentally induced in the dentate gyrus and are propagated into the hippocampus proper.

Animals↗

PubMed2002

Effects of barium, furosemide, ouabaine and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on ionophoretically-induced changes in extracellular potassium concentration in hippocampal slices from rats and from patients with epilepsy.

Glial cells limit local K(+)-accumulation by K(+)-uptake through different mechanisms, sensitive to Ba(2+), ouabaine, furosemide, or DIDS. Since the relative contribution of these mechanisms has not yet been determined, we studied the effects of bath-applied barium (2 mM), ouabaine (9 microM), furosemide (2 mM), and DIDS (1 mM) on ionophoretically-induced rises in [K(+)](o) in the pyramidal layer of area CA1 from normal rat slices, in the presence of glutamate receptor (Glu-R) antagonists. We also investigated the effect of barium on ionophoretically-induced tetrapropylammonium (TPA(+))-signals in order to test for barium-induced changes of the extracellular space. Finally, we repeated the barium experiment on slices from human non-sclerotic and sclerotic hippocampal specimens to assess a reduced glial capability for barium-sensitive K(+)-uptake in sclerotic tissue from epilepsy patients. In normal rat slices barium augmented ionophoretically-induced rises in [K(+)](o) by approximately 120%, also in the presence of tetrodotoxin (TTX) (by approximately 150%), but did not significantly affect the TPA(+)-signal. Ouabaine also augmented the K(+)-signal, but only by 27%. Furosemide and DIDS had negligible effects. In slices from sclerotic human hippocampus an augmentation of the K(+)-signal by barium was absent. Thus barium augments ionophoretically-induced K(+)-signals to a similar extent as previously shown for stimulus-induced signals. We suggest that glial barium-sensitive K(+)-buffer mechanisms reduce fast local rises of [K(+)](o) by at least 50%. This capability of glial cells is extremely reduced in area CA1 of slices from human sclerotic hippocampal specimens.

2-Amino-5-phosphonovalerate↗

PubMed2002

Nitric oxide modulates low-Mg2+-induced epileptiform activity in rat hippocampal-entorhinal cortex slices.

The production of nitric oxide (NO) during low-Mg2+-induced epileptiform activity in rat hippocampal-entorhinal cortex slices was investigated by real-time monitoring using 1,2-diaminoanthraquinone (DAQ). NO reacts with the aromatic amino groups of DAQ at neutral pH and in the presence of oxygen to form the fluorescence product 1H-anthra-[1,2d]-[1,2,3]triazole-6,11-dione (ATD). The DAQ-induced formation of ATD required NO and was insensitive to radical oxygen species. Removal of Mg2+ ions from the artificial cerebrospinal fluid (ACSF) induced a significant elevation in the ATD fluorescence signal. The application of L-arginine (2 mM), a substrate of nitric oxide synthase (NOS), caused a comparable increase in the ATD fluorescence signal. Furthermore, ATD signal increase induced either by low-Mg2+ ACSF or by L-arginine was sensitive to N-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor. The application of L-NAME (200 microM) caused a complete blockade of low-Mg2+-induced epileptiform activity. Under this condition, increasing NO concentration by addition of the NO donor S-nitroso-N-acetylpenicillamine (200 microM) reinduced the epileptiform activity. It has been concluded that onset and maintenance of low-Mg2+-induced spontaneous epileptiform activity are modulated by NO concentration. Further NO imaging studies may help to elucidate the role of NO in detail and may bring to light new means for epilepsy therapy.

Animals↗

PubMed2002

Spatial nitric oxide imaging using 1,2-diaminoanthraquinone to investigate the involvement of nitric oxide in long-term potentiation in rat brain slices.

Long-term potentiation (LTP), a model of activity-dependent synaptic plasticity, involves the persistent enhancement of excitatory neurotransmission. Several recent studies have suggested a critical role for nitric oxide (NO) production in hippocampal LTP. However, increase in NO production in living tissue has not yet been directly demonstrated. We used 1,2-diaminoanthraquinone (DAQ) to demonstrate NO production in rat brain slices in relation to induction of LTP. DAQ was found to be without neurotoxic effects and it neither influenced normal evoked field potential amplitudes nor did it affect induction of LTP in comparison to controls. We found that DAQ-induced fluorescence is elevated within a limited area of about 40,000 microm(2) during LTP induction in the hippocampal area CA1. Furthermore, we could demonstrate that application of the NO-synthetase inhibitor l-NAME inhibits the induction of LTP in area CA1 and causes a strong reduction of DAQ induced fluorescence. Our results are consistent with the hypothesis that NO can serve as a retrograde messenger during induction of LTP in the hippocampus.

Animals↗

PubMed2002

Kindling enhances kainate receptor-mediated depression of GABAergic inhibition in rat granule cells.

Several lines of evidence indicate a substantial contribution of kainate receptors to temporal lobe seizures. The activation of kainate receptors located on hippocampal inhibitory interneurons was shown to reduce GABA release. A reduced GABA release secondary to kainate receptor activation could contribute to an enhanced seizure susceptibility. As the dentate gyrus serves a pivotal gating function in the spread of limbic seizures, we tested the role of kainate receptors in the regulation of GABA release in the dentate gyrus of control and kindled animals. Application of glutamate (100 micro m) in the presence of the NMDA receptor antagonist d-APV and the AMPA receptor antagonist, SYM 2206 caused a slight depression of evoked monosynaptic inhibitory postsynaptic currents (IPSCs) in control, but a substantial decrease in kindled dentate granule cells. The observation that kainate receptor activation altered paired-pulse depression and reduced the frequency of TTX-insensitive miniature IPSCs without affecting their amplitude is consistent with a presynaptic action on the inhibitory terminal to reduce GABA release. In kindled preparations, neither glutamate (100 micro m) nor kainate (10 micro m) applied in a concentration known to depolarize hippocampal interneurons led to an increase of the TTX-sensitive spontaneous IPSC frequency nor to changes of the postsynaptic membrane properties. Consistently, the inhibitory effect on evoked IPSCs was not affected by the presence of the GABAB receptor antagonist, CGP55845A, thus excluding a depression by an enhanced release of GABA acting on presynaptic GABAB receptors. The enhanced inhibition of GABA release following presynaptic kainate receptor activation favours a use-dependent hyperexcitability in the epileptic dentate gyrus.

Animals↗

PubMed2002

Differential excitability and voltage-dependent Ca2+ signalling in two types of medial entorhinal cortex layer V neurons.

The entorhinal cortex (EC) is a key structure in memory formation, relaying sensory information to the hippocampal formation and processed information to the neocortex. EC neurons in the deep layers modulate the transfer of sensory information by the superficial layers and the dentate gyrus, and form the output to the neocortex. Here we characterize two types of EC layer V neurons by their fluorescence morphology, electrophysiology and intracellular Ca2+ signalling using intracellular recording and Ca2+ imaging. Pyramidal neurons show, in response to depolarizing current pulses, regular firing with strong adaptation and a fast and medium afterhyperpolarization (AHP) which are separated by a depolarizing notch and, with hyperpolarizing current injection, a transient sag. Multipolar cells respond to depolarization with delayed firing with very weak adaptation and have no depolarizing notch between fast and medium AHP and no sag with hyperpolarization. The delayed firing was blocked by 30 micro m 4-aminopyridine, indicating mediation by the D-type potassium current. Subthreshold depolarization evoked membrane potential oscillations of 2-5 Hz in both cell types and an increase in [Ca2+]i of 37 nm in pyramidal and 59 nm in multipolar neurons. Repetitive firing at 10 Hz for 30 s increased [Ca2+]i in pyramidal and multipolar neurons by 194 and 295 nm, respectively. Differential temporal firing and Ca2+ signalling suggest specific information processing and synaptic memory storage possibilities in these two layer V cell types of the EC.

4-Aminopyridine↗

PubMed2002

Coupling of electrical and metabolic activity during epileptiform discharges.

Changes in electrical activity, ionic microenvironments, and intracellular Ca concentration were measured during recurrent seizures induced by low Mg in slices and slice cultures. In both preparations, initial seizure-like events (SLEs) changed after some time into drug-refractory late recurrent discharges. In slice cultures, there was considerable cell loss in all hippocampal areas after 2 h of status epilepticus. During recurrent SLEs, the NAD(P)H autofluorescence declined, as did intramitochondrial calcium signals, indicating mitochondrial damage. At the same time, ethidium signals indicated increased radical oxygen species production. These alterations could be reduced by alpha-tocopherol, which also protected slice cultures against status epilepticus-induced cell death.

Animals↗

PubMed2002

Endothelin-1 potently induces Leão's cortical spreading depression in vivo in the rat: a model for an endothelial trigger of migrainous aura?

According to the 'neuronal' theory, cortical spreading depression (CSD) is the pathophysiological correlate of migrainous aura. However, the 'vascular' theory has implicated altered vascular function in the induction of aura symptoms. The possibility of a vascular origin of aura symptoms is supported, e.g. by the clinical observation that cerebral angiography frequently provokes migrainous aura. This suggests that endothelial irritation may somehow initiate one of the pathways resulting in migrainous aura. Up to now, an endothelium-derived factor has never been shown to trigger CSD. Here, for the first time, we demonstrate and characterize the ability of the vasoconstrictor and astroglial/neuronal modulator endothelin-1 to trigger Leão's 'spreading depression of activity' in vivo in rats. At a concentration range between 10 nM and 1 microM, endothelin-1 induced changes characteristic of CSD with regard to the rate of propagation, steady (direct current) potential and extracellular K(+)-concentration. A spreading hyperaemia followed by oligaemia was observed similar to those in K(+)-induced CSD. Endothelin-1 did not provoke changes characteristic of a terminal depolarization. The mechanism by which endothelin-1 generated CSD involved the N-methyl-D-asparate receptor. Cerebral blood flow decreased slightly, but significantly, before endothelin-1 generated CSD. A vasodilator (NO*-donor) shifted the threshold for CSD induction to higher concentrations of endothelin-1. Endothelin-1, in contrast to K(+), did not induce CSD in rat brain slices suggesting indirectly that endothelin-1 may require intact perfusion to exert its effects. In conclusion, endothelin-1 was found in the experiment to be the most potent inducer of CSD currently known. We propose endothelin-1 as a possible candidate for the yet enigmatic link between endothelial irritation and migrainous aura.

Animals↗