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Biomedical subjects

U Heinemann

Publications and source records attributed to U Heinemann.

At least 37 records · Page 2Linked to original sources
PubMed2001

Enhanced spontaneous transmitter release is the earliest consequence of neocortical hypoxia that can explain the disruption of normal circuit function.

After the onset of an acute episode of arrested circulation to the brain and consequent cerebral hypoxia, EEG changes and modifications of consciousness ensue within seconds. This in part reflects the rapid effect of hypoxia on the neocortex, where oxygen deprivation leads to impaired neuronal excitability and abnormal synaptic transmission. To identify the cellular mechanisms responsible for the earliest changes in neocortical function and to determine their time course, we have used patch-in-slice recording techniques to investigate the effects of acute hypoxia on the synaptic and intrinsic properties of layer 5 neurons. Coronal slices of mouse somatosensory cortex were maintained at 37 degrees C and challenged with episodes of hypoxia (3-4 min of exposure to 95% N(2), 5% CO(2)). In recordings with cell-attached patch electrodes, activation of ATP-sensitive potassium channels first became detectable 211 +/- 11 sec (range, 185-240 sec; n = 6 patches) after the onset of hypoxia. Similar recording techniques revealed no alterations in the properties of Na(+) currents in the first 4 min after the onset of hypoxia. The earliest hypoxia-induced disturbance was a marked increase in the frequency of spontaneous EPSCs and IPSCs, which began within 15-30 sec of the removal of oxygen. This rapid synaptic effect was not sensitive to TTX and was present in Ca(2+)-free perfusate, indicating that the hypoxia had a direct influence on the vesicular release mechanisms. The incoherent, massive increase in miniature PSCs would be expected to deplete the readily releasable pool of vesicles in cortical terminals, and to thereby markedly distort the neuronal interactions that underlie normal circuit function.

Adenosine Triphosphate↗
PubMed2001

Entorhinal cortex projection cells to the hippocampal formation in vitro.

Retrogradely labeled cells in superficial and deep layers of the entorhinal cortex (EC) were analyzed following application of the fluorescent tracer rhodamine-dextran-amine in different sites of the hippocampal formation in a slice preparation. The results demonstrate a strong projection from layer IV/V to the dentate gyrus, that is in slices significantly stronger than that from layer II. In deep layers a large number of multipolar cells were found which were only labeled by dye application to the subiculum. Patch-clamp recordings from these cells revealed intrinsic low threshold membrane potential oscillations, suggesting their possible contribution to oscillatory network activity of the EC and subiculum.

Animals↗
PubMed2001

Mild postischemic hypothermia is neuroprotective in the immature rat neocortex slice.

Mild hypothermia as an intervention after perinatal asphyxia may prevent neurological damage in the newborn. We used stimulus-induced field potentials to monitor recovery from oxygen and glucose deprivation (OGD) in neocortex slices of 6-8-day-old wistar rats. OGD after a latency of 10.7+/-2.1 min (mean+/-S.E.) resulted in an anoxic depolarisation with an amplitude of 5.4+/-2.4 mV. Mild hypothermia of 31 degrees C (vs. 35 degrees C in the control group) was applied for 60 min after end of OGD. The 20, 40, 60 and 80% recovery of the field potential amplitude was significantly faster in the hypothermia group in comparison to the control group. These data indicate that mild postischemic hypothermia may have neuroprotective effects after perinatal asphyxia.

Animals↗
PubMed2001

Molecular genetic alterations on chromosomes 11 and 22 in ependymomas.

Ependymomas arise from the ependymal cells at different locations throughout the brain and spinal cord. These tumors have a broad age distribution with a range from less than 1 year to more than 80 years. In some intramedullary spinal ependymomas, mutations in the neurofibromatosis 2 (NF2) gene and loss of heterozygosity (LOH) on chromosome arm 22q have been described. Cytogenetic studies have also identified alterations involving chromosome arm 11q, including rearrangements at 11q13, in ependymomas. We analyzed 21 intramedullary spinal, 14 ventricular, 11 filum terminale and 6 intracerebral ependymomas for mutations in the MEN1 gene, which is located at 11q13, and mutations in the NF2 gene, which is located at 22q12, as well as for LOH on 11q and 22q. NF2 mutations were found in 6 tumors, all of which were intramedullary spinal and all of which displayed LOH 22q. Allelic loss on 22q was found in 20 cases and was significantly more frequent in intramedullary spinal ependymomas than in tumors in other locations. LOH 11q was found in 7 patients and exhibited a highly significant inverse association with LOH 22q (p<0.001). A hemizygous MEN1 mutation was identified in 3 tumors, all of which were recurrences from the same patient. Interestingly, the initial tumor corresponded to WHO grade II and displayed LOH 11q but not yet a MEN1 mutation. In 2 subsequent recurrences, the tumor had progressed to anaplastic ependymoma (WHO grade III) and exhibited a nonsense mutation in exon 10 of MEN1 (W471X) in conjunction with LOH 11q. This suggests that loss of wild-type MEN1 may be involved in the malignant progression of a subset of ependymomas. To conclude, our findings provide evidence for different genetic pathways involved in ependymoma formation and progression, which may allow to define genetically and clinically distinct tumor entities.

Adolescent↗
PubMed2001

Ischaemia triggered by spreading neuronal activation is inhibited by vasodilators in rats.

It has been previously shown that spreading neuronal activation can generate a cortical spreading ischaemia (CSI) in rats. The purpose of the present study was to investigate whether vasodilators cause CSI to revert to a normal cortical spreading depression (CSD).A KCl-induced CSD travelled from an open cranial window to a closed window where the cortex was superfused with physiological artificial cerebrospinal fluid (ACSF). At the closed window, recordings revealed a short-lasting negative slow potential shift accompanied by a variable, small and short initial hypoperfusion followed by hyperaemia and then oligaemia. In contrast, spreading neuronal activation locally induced CSI at the closed window when ACSF contained a NO. synthase (NOS) inhibitor, N(G)-nitro-L-arginine, and an increased K+ concentration ([K+]ACSF). CSI was characterised by a sharp and prolonged initial cerebral blood flow decrease to 29 +/- 11 % of the baseline and a prolonged negative potential shift. Co-application of a NOá donor, S-nitroso-N-acetylpenicillamine, and NOS inhibitor with high [K+]ACSF re-established a short-lasting negative potential shift and spreading hyperaemia typical of CSD. Similarly, the NO.-independent vasodilator papaverine caused CSI to revert to a pattern characteristic of CSD. In acute rat brain slices, NOS inhibition and high [K+]ACSF did not prolong the negative slow potential shift compared to that induced by high [K+]ACSF alone. The data indicate that the delayed recovery of the slow potential was caused by vasoconstriction during application of high [K+]ACSF and a NOS inhibitor in vivo. This supports the possibility of a vicious circle: spreading neuronal activation induces vasoconstriction, and vasoconstriction prevents repolarisation during CSI. Speculatively, this pathogenetic process could be involved in migraine-induced stroke.

Animals↗
PubMed2001

High oxygen tension leads to acute cell death in organotypic hippocampal slice cultures.

Increased oxygen tension in the central nervous system can be of relevance in different clinical situations, e.g. hyperbaric oxygen treatment during resuscitation of newborns in asphyxia as well as during seizures in children and adults where the supply of oxygen to tissue is increased by elevated cerebral blood flow. We focused on changes in neuronal tissue by investigating the impact of different oxygen tensions on juvenile rat hippocampal slice cultures using extracellular field potential recordings and propidium iodide (PI) staining for cell death determination. Slice cultures were prepared following the Stoppini technique (postnatal days 6-8). Electrophysiological responses in area CA1 to hilar stimulation were recorded every 15 min after an initial equilibration period of 60 min. Slice cultures maintained in 95% oxygen showed a 53% (S.E.M.=17%; n=10) run-down in amplitudes of the evoked responses over the observation time course of 90 min. In contrast, slice cultures maintained in 19% oxygen showed no run-down in amplitudes (S.E.M.=9%; n=18). PI staining of the slice cultures carried out immediately after the electrophysiological measurements indicated a dramatic cell death rate in the high oxygen tension group compared to those maintained in 19% oxygen. Interestingly, epileptiform activity (seizure-like events, spreading depression-like events) occurred in some slice cultures dependent on oxygen tension. Altered paired-pulse index of evoked responses suggests a loss of GABAergic function, especially in the 95% oxygen tension group. These results demonstrate a high sensitivity to oxygen in juvenile rat hippocampal slice cultures, in contrast to acutely prepared juvenile and adult rat hippocampal slices.

Animals↗
PubMed2001

High-throughput three-dimensional protein structure determination.

In the wake of finished genomic sequencing projects, high-throughput analysis techniques are being developed in various fields of functional genomics. Of special interest in this regard is the three-dimensional structure analysis of proteins by X-ray crystallography and NMR spectroscopy, which has been characterized by distinctly low-throughput in the past. A number of recent advances in instrumentation and software are promising to radically change this situation, leaving the production of suitable protein samples as the sole rate-limiting step in structural analyses.

Crystallography, X-Ray↗
PubMed2001

Monitoring NAD(P)H autofluorescence to assess mitochondrial metabolic functions in rat hippocampal-entorhinal cortex slices.

Changes in neuronal energy metabolism, mitochondrial functions and homeostasis of reactive oxygen species are often supposed to induce alterations in neuronal activity in hippocampal slice models. In order to investigate the NAD(P)H autofluorescence signal in brain slice models, methods to monitor NAD(P)H signal in isolated mitochondria as described by Chance et al. [J. Biol. Chem. 254 (1979) 4764] and dissociated neurons as described by Duchen [Biochem. J. 283 (1992) 41] were adapted to recording conditions required for brain slices. Considering different experimental questions, we established an approach to monitor NAD(P)H autofluorescence signals from hippocampal slices of 400 microm thickness under either submerged or interface conditions. Therefore the procedure described here allows the measurement of NAD(P)H autofluorescence under conditions typically required in electrophysiological experiments. Depolarization of plasma membrane caused by electrical stimulation or application of glutamate (100 microM) resulted in a characteristic initial decrease followed by a long-lasting increase in the NAD(P)H autofluorescence signal. H(2)O(2) (100 microM) evoked a strong NAD(P)H signal decrease indicating direct oxidation to the nonfluorescencend NAD(P)(+). In contrast, the increase in NAD(P)H signal that followed a brief inhibition of mitochondrial respiratory chain complex I using rotenone (1 microM) indicated an accumulation of NAD(P)H. However, in presence of rotenone (1 microM) electrically evoked long-lasting NAD(P)H signal overshoot decreased progressively, due to a negative feedback of accumulated NAD(P)H to the citrate cycle. A comparable reduction in NAD(P)H signal increase were observed during low-Mg(2+) induced epileptiform activity, indicating a relative energy failure. In conclusion, the method presented here allows to monitor NAD(P)H autofluorescence signals to gain insight into the coupling of neuronal activity, energy metabolism and mitochondrial function in brain slice models.

Animals↗
PubMed2001

Structure of the DLM-1-Z-DNA complex reveals a conserved family of Z-DNA-binding proteins.

The first crystal structure of a protein, the Z alpha high affinity binding domain of the RNA editing enzyme ADAR1, bound to left-handed Z-DNA was recently described. The essential set of residues determined from this structure to be critical for Z-DNA recognition was used to search the database for other proteins with the potential for Z-DNA binding. We found that the tumor-associated protein DLM-1 contains a domain with remarkable sequence similarities to Z alpha(ADAR). Here we report the crystal structure of this DLM-1 domain bound to left-handed Z-DNA at 1.85 A resolution. Comparison of Z-DNA binding by DLM-1 and ADAR1 reveals a common structure-specific recognition core within the binding domain. However, the domains differ in certain residues peripheral to the protein-DNA interface. These structures reveal a general mechanism of Z-DNA recognition, suggesting the existence of a family of winged-helix proteins sharing a common Z-DNA binding motif.

Adenosine Deaminase↗
PubMed2001

Properties of entorhinal cortex deep layer neurons projecting to the rat dentate gyrus.

Medial entorhinal cortex (EC) deep layer neurons projecting to the dentate gyrus (DG) were studied. Neurons, retrogradely-labelled with rhodamine-dextran-amine were characterized electrophysiologically with the patch clamp technique and finally labelled with biocytin. Pyramidal and nonpyramidal neurons form projections from the deep layers of the EC to the molecular layer of the DG. In addition, both classes of projection neurons send ascending axon collaterals to the superficial layers of the EC. Both classes of neurons were characterized physiologically by regular action potential firing upon depolarizing current injection. While a substantial number of pyramidal projection cells showed intrinsic membrane potential oscillations, none of the studied nonpyramidal cells exhibited oscillations. Despite the morphological similarity of bipolar and multipolar cells to those of GABAergic interneurons in the EC, their electrophysiological characteristics were similar to those of principal neurons and immunocytochemistry for GABA was negative. We conclude, that neurons of the deep layers of the medial EC projecting to the DG may function as both local circuit and projecting neurons thereby contributing to synchronization between deep layers of the EC, superficial layers of the EC and the DG.

Animals↗
PubMed2001

Ca2+ signalling and changes of mitochondrial function during low-Mg2+-induced epileptiform activity in organotypic hippocampal slice cultures.

Several lines of evidence indicate that augmented neuronal activity is associated with increased mitochondrial function, however, the mechanisms of coupling are still unclear. In this study we used a low extracellular Mg2+ concentration and short stimulus trains to evoke neuronal hyperactivity in the form of seizure-like events (SLE) in hippocampal slice cultures. Simultaneous microfluorimetric and electrophysiological techniques were applied to gain insight into changes of Ca2+ concentration in different compartments and into mitochondrial function. SLEs were associated with a large decrease of the extracellular Ca2+ concentration ([Ca2+]e), a spiking increase of the cytoplasmic and a smoothed elevation of the mitochondrial Ca2+ concentration (cytoplasmic concentration [Ca2+]i; intramitrochondrial concentration [Ca2+]m). Following an initial apparent decline in the mitochondrial membrane potential (DeltaPsi) and NAD(P)H autofluorescence, mitochondria depolarized and NADH production was augmented. Furthermore, SLEs were associated with increased oxidation of dihydroethidine (HEt). Our data suggest that intramitochondrial Ca2+ accumulation stimulates NADH production and production of radical oxygen species (ROS). Interestingly, mitochondrial depolarization followed [Ca2+]i and [Ca2+]m changes with a delay implying that electrogenic extrusion of Ca2+ from the mitochondrial matrix might be responsible for the depolarization of the mitochondrial membrane.

Animals↗
PubMed2001

Kindling induces transient fast inhibition in the dentate gyrus--CA3 projection.

The granule cells of the dentate gyrus (DG) send a strong glutamatergic projection, the mossy fibre tract, toward the hippocampal CA3 field, where it excites pyramidal cells and neighbouring inhibitory interneurons. Despite their excitatory nature, granule cells contain small amounts of GAD (glutamate decarboxylase), the main synthetic enzyme for the inhibitory transmitter GABA. Chronic temporal lobe epilepsy results in transient upregulation of GAD and GABA in granule cells, giving rise to the speculation that following overexcitation, mossy fibres exert an inhibitory effect by release of GABA. We therefore stimulated the DG and recorded synaptic potentials from CA3 pyramidal cells in brain slices from kindled and control rats. In both preparations, DG stimulation caused excitatory postsynaptic potential (EPSP)/inhibitory postsynaptic potential (IPSP) sequences. These potentials could be completely blocked by glutamate receptor antagonists in control rats, while in the kindled rats, a bicuculline-sensitive fast IPSP remained, with an onset latency similar to that of the control EPSP. Interestingly, this IPSP disappeared 1 month after the last seizure. When synaptic responses were evoked by high-frequency stimulation, EPSPs in normal rats readily summate to evoke action potentials. In slices from kindled rats, a summation of IPSPs overrides that of the EPSPs and reduces the probability of evoking action potentials. Our data show for the first time that kindling induces functionally relevant activity-dependent expression of fast inhibition onto pyramidal cells, coming from the DG, that can limit CA3 excitation in a frequency-dependent manner.

2-Amino-5-phosphonovalerate↗
PubMed2001

Fluorescent tracer in pilocarpine-treated rats shows widespread aberrant hippocampal neuronal connectivity.

Neuronal fibres of the hippocampal formation of normal and chronic epileptic rats were investigated by fluorescent tracing methods using the pilocarpine model of limbic epilepsy. Two months after onset of spontaneous limbic seizures, hippocampal slices were prepared and maintained in vitro for 10 h. Small crystals of fluorescent dye [fluorescein (fluoro-emerald) and tetramethylrhodamine (fluoro-ruby)] were applied to different hippocampal regions. The main findings were: (i) in control rats there was no supragranular labelling when the mossy fibre tract was stained in stratum radiatum of area CA3. However, in epileptic rats a fibre network in the inner molecular layer of the dentate gyrus was retrogradely labelled; (ii) a retrograde innervation of area CA3 by CA1 pyramidal cells was disclosed by labelling remote CA1 neurons after dye injection into the stratum radiatum of area CA3 in chronic epileptic rats; (iii) labelling of CA1 neurons apart from the injection site within area CA1 was observed in epileptic rats but not in control animals; and (iv), a subicular-hippocampal projection was present in pilocarpine-treated rats when the tracer was injected just below the stratum pyramidale of area CA1. The findings show that fibre rearrangement in distinct regions of the epileptic hippocampal formation can occur as an aftermath of pilocarpine-induced status epilepticus.

Action Potentials↗
PubMed2001

Astrocyte-released cytokines induce ramification and outward K+ channel expression in microglia via distinct signalling pathways.

Differentiation of microglial cells is characterized by transformation from ameboid into ramified cell shape and up-regulation of K+ channels. The processes of microglial differentiation are controlled by astrocytic factors. The mechanisms by which astrocytes cause developmental changes in morphological and electrophysiological properties of microglia have remained unclear. We show here that the cytokines transforming growth factor-beta (TGF-beta), macrophage colony-stimulating factor (M-CSF) and granulocyte/macrophage colony-stimulating factor (GM-CSF) are released by astrocytes at concentrations sufficient to induce ramification and up-regulation of delayed rectifier (DR) K+ channels in microglia. Transformation from ameboid into ramified morphology induced in microglia by exposure to astrocyte-conditioned medium (ACM) was inhibited by neutralizing antibodies against TGF-beta, M-CSF or GM-CSF, whilst ACM-induced DR channel expression was exclusively inhibited by antibodies against TGF-beta. Although both ramification and DR channel up-regulation occurred simultaneously, DR channel blockade by charybdotoxin failed to inhibit microglial ramification. The ACM-induced ramification of microglia was inhibited by the tyrosine kinase inhibitor genistein, whereas DR channel up-regulation did not occur in the presence of the serine/threonine kinase inhibitor H7. Our data suggest that astrocytes modulate processes of microglial differentiation in parallel but via distinct signalling pathways.

Animals↗
PubMed2001

Activity-dependent changes of the presynaptic synaptophysin-synaptobrevin complex in adult rat brain.

The vesicular protein synaptobrevin contributes to two mutually exclusive complexes in mature synapses. Synaptobrevin tightly interacts with the plasma membrane proteins syntaxin and SNAP 25 forming the SNARE complex as a prerequisite for exocytotic membrane fusion. Alternatively, synaptobrevin binds to the vesicular protein synaptophysin. It is unclear whether SNARE complex formation is diminished or facilitated when synaptobrevin is bound to synaptophysin. Here we show that the synaptophysin-synaptobrevin complex is increased in adult rat brain after repeated synaptic hyperactivity in the kindling model of epilepsy. Two days after the last kindling-induced stage V seizure the relative amount of synaptophysin-synaptobrevin complex obtained by co-immunoprecipitation from cortical and hippocampal membranes was increased twofold compared to controls. By contrast the relative amounts of various synaptic proteins as well as that of the SNARE complex did not change in membrane preparations from kindled rats compared to controls. The increased amount of synaptophysin-synaptobrevin complex in kindled rats supports the idea that this complex represents a reserve pool for synaptobrevin enabling synaptic vesicles to adjust to an increased demand for synaptic efficiency. We conclude that the synaptophysin-synaptobrevin interaction is involved in activity-dependent plastic changes in adult rat brain.

Age Factors↗
PubMed2001

Protein crystallography with neutrons--status and perspectives.

The current state of the art and future perspectives for protein crystallography with neutrons have been reviewed and discussed at an international workshop recently organized by the Hahn-Meitner-Institut (HMI) and Max-Delbrück-Center (MDC) in Berlin, and supported by the Neutron Round Table activity. The main topics discussed and the conclusions drawn are presented in this paper. While the available flux from even the most powerful neutron sources is much lower than that available at a modern synchrotron-radiation source, there is no doubt that neutrons can additionally provide vital information in structural biology that is difficult to obtain using other methods. One of the overriding themes at the workshop was the fact that the current instrumentation and facilities do not fully exploit the neutron beams that are available. Current trends indicate that many of the problems that have limited the efficiency of biological neutron scattering have been recognized and with new technological advances important efforts are under way to tackle these problems. It is clear that the use of neutrons in biology will become increasingly significant at existing and new neutron sources.

Crystallography, X-Ray↗
PubMed2001

Enhanced temporal stability of cholinergic hippocampal gamma oscillations following respiratory alkalosis in vitro.

The decrease in brain CO(2) partial pressure (pCO(2)) that takes place both during voluntary and during pathological hyperventilation is known to induce gross alterations in cortical functions that lead to subjective sensations and altered states of consciousness. The mechanisms that mediate the effects of the decrease in pCO(2) at the neuronal network level are largely unexplored. In the present work, the modulation of gamma oscillations by hypocapnia was studied in rat hippocampal slices. Field potential oscillations were induced by the cholinergic agonist carbachol under an N-methyl-D-aspartate (NMDA)-receptor blockade and were recorded in the dendritic layer of the CA3 region with parallel measurements of changes in interstitial and intraneuronal pH (pH(o) and pH(i), respectively). Hypocapnia from 5 to 1% CO(2) led to a stable monophasic increase of 0.5 and 0.2 units in pH(o) and pH(i), respectively. The mean oscillation frequency increased slightly but significantly from 32 to 34 Hz and the mean gamma-band amplitude (20 to 80 Hz) decreased by 20%. Hypocapnia induced a dramatic enhancement of the temporal stability of the oscillations, as was indicated by a two-fold increase in the exponential decay time constant fitted to the autocorrelogram. A rise in pH(i) evoked by the weak base trimethylamine (TriMA) was associated with a slight increase in oscillation frequency (37 to 39 Hz) and a decrease in amplitude (30%). Temporal stability, on the other hand, was decreased by TriMA, which suggests that its enhancement in 1% CO(2) was related to the rise in pH(o). In 1% CO(2), the decay-time constant of the evoked monosynaptic pyramidal inhibitory postsynaptic current (IPSC) was unaltered but its amplitude was enhanced. This increase in IPSC amplitude seems to significantly contribute to the enhancement of temporal stability because the enhancement was almost fully reversed by a low concentration of bicuculline. These results suggest that changes in brain pCO(2) can have a strong influence on the temporal modulation of gamma rhythms.

2-Amino-5-phosphonovalerate↗