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Biomedical subjects

U Heinemann

Publications and source records attributed to U Heinemann.

At least 199 records · Page 11Linked to original sources
PubMed1995

Serotonin reduces synaptic excitation of principal cells in the superficial layers of rat hippocampal-entorhinal cortex combined slices.

The cells of the entorhinal cortex receive a dense innervation of serotonergic fibres from the Raphe nuclei and express a high density of 5-hydroxytryptamine 1A (5-HT1A) receptors. We investigated the effects of serotonin on excitatory synaptic transmission in principal cells from entorhinal cortex layers II and III within hippocampal-entorhinal cortex combined slices. Although serotonin had an effect upon the membrane conductance of some, but not all cells, its most pronounced action was to reduce stimulus evoked excitatory synaptic potentials and currents (EPSP/Cs). Both alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and N-methyl-D-aspartate receptor-mediated EPSPs were reduced to similar extents over a range of concentrations. Since the principal cells in layer II and layer III are the main projection cells of the entorhinal cortex, these inhibitory effects of serotonin may have implications for the transfer of information to the hippocampus.

Action Potentials↗
PubMed1995

Crystal structure and site-directed mutagenesis of Bacillus macerans endo-1,3-1,4-beta-glucanase.

In beta-glucans those beta-1,4 glycosidic bonds which are adjacent to beta-1,3 bonds are cleaved by endo-1,3-1,4-beta-glucanases (beta-glucanases). Here, the relationship between structure and activity of the beta-glucanase of Bacillus macerans is studied by x-ray crystallography and site-directed mutagenesis of active site residues. Crystal structure analysis at 2.3-A resolution reveals a jelly-roll protein structure with a deep active site channel harboring the amino acid residues Trp101, Glu103, Asp105, and Glu107 as in the hybrid Bacillus beta-glucanase H(A16-M) (Keitel, T., Simon, O., Borriss, R., and Heinemann, U. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5287-5291). Different mutant proteins with substitutions in these residues are generated by site-directed mutagenesis, isolated, and characterized. Compared with the wild-type enzyme their activity is reduced to less than 1%. Several mutants with isosteric substitutions in Glu103 and Glu107 are completely inactive, suggesting a direct role of these residues in glycosyl bond hydrolysis. The kinetic properties of mutant beta-glucanases and the crystal structure of the wild-type enzyme are consistent with a mechanism where Glu103 and Glu107 are the catalytic amino acid residues responsible for cleavage of the beta-1,4 glycosidic bond within the substrate molecule.

Amino Acid Sequence↗
PubMed1995

Effects of low glucose levels on changes in [Ca2+]o induced by stimulation of Schaffer collaterals under conditions of blocked chemical synaptic transmission in rat hippocampal slices.

It has been shown previously that hypoxia reduced presumed presynaptic Ca2+ entry in area CA1 of rat hippocampal slices, perhaps due to energy depletion in presynaptic endings. This would imply that hypoglycaemia also affects presynaptic Ca2+ uptake. We therefore studied effects of glucose withdrawal on stimulus induced changes in [Ca2+]o in area CA1 of rat hippocampal slices. After blockade of synaptic transmission by application of the glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 30 microM) and L-(+)-2-amino-5-phosphonovaleric acid (2APV, 30 microM) stimulation of Schaffer collaterals resulted in small but significant reductions in [Ca2+]o which could be attributed to presynaptic Ca2+ entry. Stimulation of the alveus resulted in somewhat reduced Ca2+ signals mediated to a large extent by Ca2+ entry into dendrites and somatas of pyramidal cells. Removal of glucose results in an augmentation of the presumed presynaptic Ca2+ entry by a factor of 2-3. Decreases in [Ca2+]o induced by repetitive stimulation of the alveus were initially augmented and then disappeared presumably due to irreversible damage of pyramidal neurones. This finding suggests that depletion of ATP alone does not account for the depressive effect of hypoxia on presynaptic Ca2+ entry.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1995

Ischaemia-induced long-term hyperexcitability in rat neocortex.

The long-term structural and functional consequences of transient forebrain ischaemia were studied with morphological, immunohistochemical and in vitro electrophysiological techniques in the primary somatosensory cortex of Wistar rats. After survival times of 10-17 months postischaemia, neocortical slices obtained from ischaemic animals were characterized by a pronounced neuronal hyperexcitability in comparison with untreated age-matched controls. Extra- and intracellular recordings in supragranular layers revealed all-or-none long-latency recurrent responses to orthodromic synaptic stimulation of the afferent pathway. These responses were characterized by durations up to 1.7 s, by multiple components and by repetitive synaptic burst discharges. The reversible blockade of this late activity by DL-amino-phosphonovaleric acid (APV) suggested that this activity was mediated by N-methyl-D-aspartate (NMDA) receptors. The peak conductance of inhibitory postsynaptic potentials was significantly smaller in neurons recorded in neocortical slices obtained from ischaemic animals than those from the controls. However, the average number of parvalbumin (PV)-labelled neurons per mm3, indicative of a subpopulation of GABAergic interneurons, and the average number and length of dendritic processes arising from PV-containing cells was not significantly different between ischaemic and control cortex. The prominent dysfunction of the inhibitory system in ischaemic animals occurred without obvious structural alterations in PV-labelled cells, indicating that this subpopulation of GABAergic interneurons is not principally affected by ischaemia. Our data suggest a long-term down-regulation of inhibitory function and a concurrent NMDA receptor-mediated hyperexcitability in ischaemic neocortex. These alterations may result from structural and/or functional properties of inhibitory non-PV-positive neurons or permanent functional modifications on the subcellular molecular level, i.e. alterations in the phosphorylation status of GABA and/or NMDA receptors. The net result of these long-term changes is an imbalance between the excitatory and inhibitory systems in the ischaemic cortex with the subsequent expression and manifestation of intracortical hyperexcitability.

Animals↗
PubMed1995

Effects of methysticin on three different models of seizure like events studied in rat hippocampal and entorhinal cortex slices.

Methysticin is one of the constituents of Piper methysticum which possesses anticonvulsant and neuroprotective properties. Its effects on different in vitro seizure models were tested using extracellular recordings in rat temporal cortex slices containing the hippocampus and the entorhinal cortex. Elevating [K+]0 induced seizure-like events with tonic and clonic electrographic phases in area CA1. Lowering [Ca2+]0 caused recurrent seizure like episodes with large negative field potential shifts. Lowering Mg2+ induced short recurrent discharges in area CA3 and CA1 while ictaform events lasting for many seconds were induced in the subiculum, entorhinal and temporal neocortex. In the hippocampus the activity stayed stable over a number of hours. In contrast, the ictaform events in the subiculum, entorhinal and temporal cortex changed their characteristics after one to two hours to late recurrent discharges. In a concentration-range from 10 to 100 microM methysticin reversibly blocked all these types of epileptiform activity. Decreases in [Ca2+]0 and associated slow field potentials evoked by repetitive stimulation of the stratum radiatum or the alveus remained almost unaffected by methysticin. A paired pulse stimulus paradigm used to test for effects of methysticin on synaptically evoked transient field potentials in normal medium revealed interference with mechanisms involved in frequency potentiation. While responses to alvear stimulation were largely unaffected, the responses to a paired pulse stimulus to stratum radiatum were depressed over the whole range of tested stimulus intervals. The findings suggest that methysticin has effects on different patterns of epileptiform activity possibly by interfering with processes responsible for frequency potentiation.

Animals↗
PubMed1995

Properties of voltage-gated potassium currents of microglia differentiated with granulocyte/macrophage colony-stimulating factor.

Voltage-gated whole-cell currents were recorded from cultured microglial cells which had been developed in the presence of the macrophage/microglial growth factor granulocyte/macrophage colony-stimulating factor. Outward K+ currents (IK) were most prominent in these cells. IK could be activated at potentials more positive than -40 mV. Half-maximal activation of IK was achieved at -13.8 mV and half-maximal inactivation of IK was determined at -33.8 mV. The recovery of IK from inactivation was described by a time constant of 7.9 sec. For a tenfold change in extracellular K+ concentration the reversal potential of IK shifted by 54 mV. Extracellularly applied 10 mM tetraethylammonium chloride reduced IK by about 50%, while 5 mM 4-aminopyridine almost completely abolished IK. Several divalent cations (Ba2+, Cd2+, Co2+, Zn2+) reduced current amplitudes and shifted the activation curve of IK to more positive values. Charybdotoxin (IC50 = 1.14 nM) and noxiustoxin (IC50 = 0.89 nM) blocked IK in a concentration-dependent manner, whereas dendrotoxin and mast cell degranulating peptide had no effect on the current amplitudes. The outward K+ currents showed a frequency dependence when depolarizing pulses were applied at a frequency of 1 Hz. A frequency-independent outward current (IK') characterized by the same activation behavior as IK was detected. IK' was blocked completely by 10 nM charybdotoxin or by 10 nM noxiustoxin. In contrast to its effect on IK, 10 mM tetraethylammonium chloride did not reduce IK'.

4-Aminopyridine↗
PubMed1995

Properties of voltage-gated currents of microglia developed using macrophage colony-stimulating factor.

Microglia were isolated from a murine neonatal brain cell culture in which their development had been stimulated by supplementation with the macrophage/microglial growth factor macrophage colony-stimulating factor (M-CSF). Using the whole-cell configuration of the patch-clamp technique, voltage-gated membrane currents were recorded from these microglial cells. Hyperpolarization induced inward rectifying K+ currents, as described for microglia from untreated cultures. These currents activated negative to the K+ equilibrium potential and, with a strong hyperpolarization, displayed time-dependent inactivation. The inactivation was abolished when extracellular NaCl was replaced by N-methyl-D-glucamine (NMG), thereby indicating a partial block of this K+ conductance by Na+. Inward rectifying currents were also blocked by extracellularly applied Cs+ or Ba2+. They were slightly diminished following treatment with extracellular tetraethylammonium chloride (TEA) but were not affected by 4-aminopyridine (4-AP). Upon long lasting depolarizing voltage pulses to potentials positive to 0 mV, the cells exhibited a slowly activating H+ current which could be reduced by application of inorganic polyvalent cations (Ba2+, Cd2+, Co2+, La3+, Ni2+, Zn2+) as well as by 4-AP or TEA. Based on their kinetics and pharmacological characteristics, both currents detected on M-CSF-grown microglia are suggested to correspond to the inward rectifier and the H+ current of macrophages.

Animals↗
PubMed1995

Ketamine and strychnine treatment of an infant with nonketotic hyperglycinaemia.

UNLABELLED: Non-ketotic hyperglycinaemia (NKH) is a severe seizure disorder associated with high glycine levels. Glycine is a major inhibitory neurotransmitter in the CNS, but has also modulating effects at one of the glutamate receptors, the N-methyl-D-aspartate-(NMDA) receptor. Based on this knowledge we treated a female newborn suffering from severe NKH with the NMDA receptor blocker ketamine in association with strychnine and magnesium supplementation. This treatment led to cessation of seizures, reappearance of swallowing and sucking and improved the neurological status. Some pharmacokinetic data of strychnine and ketamine in the infant are given. CONCLUSION: Ketamine in combination with strychnine may be beneficial in non-ketotic hyperglycinaemia.

Amino Acid Metabolism, Inborn Errors↗
PubMed1995

Cytokine-dependent K+ channel profile of microglia at immunologically defined functional states.

Microglia were enriched in brain cell cultures from newborn mice as a result of supplementation with the growth factors macrophage colony-stimulating factor or granulocyte/macrophage colony-stimulating factor. When separately administered these two cytokines promote the outgrowth of loosely adherent cells with similar morphology which stained positive for CD11b and nonspecific esterase. Microglial cells isolated from both types of culture were electrophysiologically characterized using the whole cell configuration of the patch-clamp technique. Different resting membrane potentials were measured. In response to hyperpolarizing and depolarizing voltage commands 68 of 91 macrophage colony-stimulating factor-cultured microglial cells exhibited only an inward rectifying potassium current. By contrast, an outward potassium current was observed on 71 of 95 granulocyte/macrophage colony-stimulating factor-grown cells. Parallel testing of their capability for antigen presentation proved the activated functional state of these microglial cells. They induce antigen-specific T cell response without prior stimulus. In comparison, cells developed with macrophage colony-stimulating factor failed to present antigen. In such resting microglia a short-term treatment with granulocyte/macrophage colony-stimulating factor or interferon-gamma provoked a strong appearance of outward potassium currents, however, only the interferon-gamma-trigger resulted in efficient antigen presentation. The differential induction of both functional parameters suggests the detection of outward potassium currents to provide an electrophysiological activation marker of microglia which is subjected to cytokine regulation but not compellingly linked to antigen presentation.

Animals↗
PubMed1995

Paroxysmal epileptiform discharges in temporal lobe slices after prolonged exposure to low magnesium are resistant to clinically used anticonvulsants.

Lowering of extracellular Mg2+ results in various forms of epileptiform activity in different parts of temporal lobe slices [5,22] which contain neocortical areas such as areas Te2 or Te3, the entorhinal cortex (EC), subiculum, hippocampal areas CA1 to CA3 and the dentate gyrus [5,11]. In the EC, the subiculum and Te2/Te3 seizure-like events (SLEs) with tonic and clonic electrographic discharge patterns, negative slow field potentials and ionic changes comparable to those during tonic-clonic seizures in intact animals were observed. After 30 to 120 min of recurrent seizure activity (80 +/- 37 min) the seizure-like events (SLEs) developed into a state of late recurrent discharges (LRDs). Since previous studies had shown that the LRDs do not respond to valproic acid in contrast to a blocking effect of this drug on SLEs, we investigated the effects of the clinically employed anticonvulsants phenytoin, carbamazepine, phenobarbital, midazolam and ethosuximide on LRDs. All these agents were unable to block the LRDs in the EC, subiculum and Te2/Te3. This was found true both for concentrations which can block SLEs and for higher concentrations. Thus we conclude that this activity may represent a model of difficult to treat status epilepticus.

Animals↗
PubMed1995

Impairment of intracortical GABAergic inhibition in a rat model of absence epilepsy.

The WAG/Rij rat strain is characterized in its EEG by the manifestation of spike-wave discharges which resemble in their spontaneous appearance and pharmacological sensitivity the absence epilepsy observed in humans. In order to test the hypothesis whether cellular intrinsic membrane and/or synaptic network properties in the neocortex are modified in this form of epilepsy, we analyzed with extra- and intracellular recording techniques the functional status of neocortical slices obtained from adult epileptic WAG/Rij rats and compared them with the data acquired from non-epileptic control Wistar rats. Intrinsic membrane properties, like resting membrane potential, neuronal input resistance and basic cellular firing characteristics, did not differ between these two strains. However, the analysis of extra- and intracellularly recorded synaptic responses revealed an intracortical hyperexcitability which was accompanied by a significant reduction in the efficiency of GABAergic inhibition. Our data indicate that the imbalance between intracortical excitatory and inhibitory mechanisms may at least contribute to the expression and augmentation of spike-wave discharges in epileptic WAG/Rij rats.

Animals↗
PubMed1995

Comparison of voltage-dependent potassium currents in rat pyramidal neurons acutely isolated from hippocampal regions CA1 and CA3.

1. The properties of voltage-gated potassium currents were studied in acutely isolated rat hippocampal pyramidal cells from area CA1 and CA3 at postnatal ages of day 6-8, 9-14, and 26-29 (P6-8, P9-14, and P26-29) with the use of the whole cell version of the patch-clamp technique. 2. The outward current pattern of all cells under investigation could be separated in a fast transient A current (IA) and a delayed rectifier-like current (IK). 3. In both preparations, IA activated and inactivated rapidly. Vh describing steady-state inactivation was -84.5 mV in CA3 cells and -85.5 mV in CA1 cells. The activation behavior was characterized by Vh = -23.8 mV in CA3 cells and -27.2 mV in CA1 cells. The removal of inactivation was monoexponential both in CA1 and CA3 neurons with time constants of 32.1 and 28.5 ms, respectively. IA was insensitive to tetraethylammonium (TEA), dendrotoxin (300 nM), and mast cell degranulating peptide (200 nM), but could be blocked with 5 mM 4-aminopyridine (4-AP) by approximately 80%. In both preparations, A currents did not depend on Ca2+ influx. 4. Delayed rectifier currents (IK) in CA1 and CA3 pyramidal neurons decayed along a double exponential time course. Steady-state inactivation was described by Vh = -79.5 mV in CA3 cells and -76.0 mV in CA1 cells. The activation curves were characterized by midpoints of -3.8 mV in CA3 cells and of -1.4 mV in CA1 cells. The removal of inactivation was monoexponential in CA1 and CA3 neurons with time constants of 210.3 and 202.4 ms, respectively. All kinetic properties were identical for the differentially decaying components of IK. In CA1 cells IK was blocked by TEA at +30 mV with an IC50 of 0.98 mM. In CA3 cells the corresponding IC50 value was 1.05 mM. About 20% of IK were insensitive to TEA. IK was partially blocked by approximately 30% with 100 microM 4-AP. Mast cell degranulating peptide (100-200 nM) and dendrotoxin (50-300 nM) had no effect on IK. 6. Perfusion of charybdotoxin (30 nM), Cd2+ (300 microM), La3+ (10 microM), or Ca(2+)-free solutions resulted in the isolation of a small noninactivating outward current component. Around 10% of IK appeared to be Ca2+ dependent in CA1 neurons. In CA3 pyramidal cells Ca(2+)-dependent outward currents seemed to be somewhat larger with approximately 20%. 7. In CA1 as well as in CA3 cells, the kinetic and pharmacological properties of IA and IK remained stable during postnatal development. However, the contribution of IA and IK to the whole cell current varied with age. IA was more prominent in CA1 cells of age group P6-8 than in age-matched CA3 cells. CA3 cells had smaller A currents and larger delayed rectifier currents than CA1 pyramidal cells. Current densities of IA and IK were analyzed during development to assess changes in the expression of these currents. With increasing postnatal age, the expression of IA was downregulated in both preparations. This effect was more pronounced in CA3 than in CA1 cells. In contrast, IK was upregulated during the same developmental period. This increase in the expression of IK was with approximately 300% much larger in CA1 cells than in CA3 cells with only approximately 50%.

4-Aminopyridine↗
PubMed1995

Serotonin reduces inhibition via 5-HT1A receptors in area CA1 of rat hippocampal slices in vitro.

We studied the effects of serotonin (5-HT) on intrinsic and synaptic responses of hippocampal CA1 cells. The effects were partially mimicked by the 5-HT1A receptor agonist, 8-OH-DPAT, and prevented by the 5-HT1A receptor antagonist, NAN-190. Polysynaptic fast and slow inhibitory postsynaptic potentials (IPSPs) were reduced in amplitude by 60-70% following application of both 5-HT and 8-OH-DPAT. Monosynaptic fast IPSPs were reduced by 60% and slow IPSPs by 90% following application of both drugs. Since there is a temporal overlap of fast and slow IPSPs, the reduction in fast IPSPs could have arisen indirectly from the larger effect of 5-HT on slow IPSPs. To overcome this problem we blocked the slow IPSPs with new, potent GABA-B antagonists, but still observed a similar reduction in the fast IPSP with 5-HT and 8-OH-DPAT. However, the reductions in the fast IPSPs could also have arisen from the 5-HT-induced total conductance increases. Using single-electrode voltage clamp and intracellular K+ channel blockers we still observed similar changes. 5-HT and 8-OH-DPAT had no effect upon GABA-A-mediated currents evoked by iontophoretic GABA application to the dendrites or the soma of CA1 pyramidal cells, Putative inhibitory internuerons were hyperpolarized by 5-HT and their evoked EPSPs strongly reduced by 5-HT and 8-OH-DPAT. Our data indicate that 5-HT modulates fast and slow synaptic inhibition of principal cells using presynaptic mechanisms involving the inhibition of inhibitory interneurons.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1994

Native-like in vivo folding of a circularly permuted jellyroll protein shown by crystal structure analysis.

A jellyroll beta-sandwich protein, the Bacillus beta-glucanase H(A16-M), is used to probe the role of N-terminal peptide regions in protein folding in vivo. A gene encoding H(A16-M) is rearranged to place residues 1-58 of the protein behind a signal peptide and residues 59-214. The rearranged gene is expressed in Escherichia coli. The resultant circularly permuted protein, cpA16M-59, is secreted into the periplasm, correctly processed, and folded into a stable and active enzyme. Crystal structure analysis at 2.0-A resolution, R = 15.3%, shows cpA16M-59 to have a three-dimensional structure nearly identical with that of the parent beta-glucanase. An analogous experiment based on the wild-type Bacillus macerans beta-glucanase, giving rise to the circularly permuted variant cpMAC-57, yields the same results. Folding of these proteins, therefore, is not a vectorial process depending on the conformation adopted by their native N-terminal oligopeptides after ribosomal synthesis and translocation through the cytoplasmic membrane.

Amino Acid Sequence↗
PubMed1994

Current density analysis of outward currents in acutely isolated rat entorhinal cortex cells.

Entorhinal cortex stellate and pyramidal cells were acutely isolated from juvenile rats. Using the whole-cell configuration of the patch-clamp method outward potassium currents were recorded. A fast transient A-current and a sustained outward current could be measured in both stellate and pyramidal cells. The A-current density was significantly larger in pyramidal cells than in stellate cells. In contrast, stellate cells possessed a larger current density of the delayed rectifier current as well as the Ca2+ activated K+ current than pyramidal cells. Moreover, superficial stellate cells exhibited large delayed rectifier currents and only small A-currents while entorhinal cortex deep layer pyramidal cells displayed larger A-currents than delayed rectifier currents.

Animals↗