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Biomedical subjects

U Heinemann

Publications and source records attributed to U Heinemann.

At least 343 records · Page 19Linked to original sources

Effects of anticonvulsants on spontaneous epileptiform activity which develops in the absence of chemical synaptic transmission in hippocampal slices.

Spontaneous epileptiform activity (SEA) develops in area CA1 of hippocampal slices, when the Ca2+ concentration in the perfusate is lowered to 0.2 mM, at which level evoked chemical synaptic transmission is blocked. We investigated the effects of different anticonvulsants on this autonomous activity, in order to determine whether the antiepileptic effect can be ascribed to an influence on neuronal excitability. Carbamazepine was the most effective to block SEA at concentrations of 1-15 microM. Phenobarbital and phenytoin depressed SEA at concentrations of 25 microM. Valproate was effective at concentrations of 2-5 mM. Midazolam, a water-soluble benzo-diazepine agonist and the N-methyl-D-aspartate antagonists, DL-alpha-aminoadipic acid and 2-amino-7-phosphonoheptanoic acid were ineffective in blocking SEA suggesting that they exert their antiepileptic action by interference with synaptic mechanisms.

Animals↗

Hippocampal slices of kindled rats reveal calcium involvement in epileptogenesis.

Daily repeated tetanic electrical stimulation (kindling) of the brain may cause a long term enhancement of synaptic transmission and epileptiform activity of progressive severity and generalisation, eventually leading to spontaneous seizures. Evidence for a cellular mechanism underlying kindling has been obtained in vitro in slices from the hippocampus of kindled rats. A marked enhancement in extracellular calcium changes, induced by electrical stimulation or by iontophoresis of excitatory aminoacids was found in kindled tissue. This implies that changes in dendritic calcium conductances are involved in kindling epileptogenesis.

Animals↗

Chemical synaptic transmission is not necessary for epileptic seizures to persist in the baboon Papio papio.

The spread and persistence of epileptic seizures have generally been attributed to chemical synaptic interactions. Using ion-sensitive microelectrodes, we showed that in the allylglycine-treated photosensitive baboon, prolonged light-induced generalized seizures were accompanied by abnormally large decreases in the concentration of extracellular calcium ions, reaching values at which chemical synaptic transmission was certainly very reduced or blocked. This feature was observed in all cortical layers. Measurements of the concentration of extracellular potassium ions in the course of such light-induced seizures indicated that the observed low values of the concentration of calcium ions could not be ascribed to the occurrence of spreading depressions. These findings showed that nonsynaptic mechanisms play a prominent role in the persistence of epileptic seizures.

Animals↗

Stimulus- and amino acid-induced calcium and potassium changes in rat neocortex.

Local changes in extracellular concentration of calcium ions, [Ca2+]o, and potassium ions, [K+]o, elicited through ionophoretic applications of the excitatory amino acids glutamate (Glu) and aspartate (Asp) or through cortical surface stimulations were measured in the rat motor cortex using paired ion-selective microelectrodes. Glu or Asp applications produced dose-dependent decreases in [Ca2+]o from a base line of 1.25 mM to as low as 0.08 mM and increases in [K+]o from 2.8 mM to as high as 13.3 mM. These ionic changes were accompanied by negative DC potential shifts of up to 15 mV. [K+]o changes were practically constant in all cortical layers, whereas [Ca2+]o changes were variable with depth, showing two localized marked maximums. One was observed in the superficial layers, 150-300 microns below cortical surface and the second at 1,100-1,300 microns, in a region corresponding roughly to layer V. Application of tetrodotoxin (TTX) onto the cortical surface abolished stimulus-evoked ionic changes, whereas amino acid-induced [Ca2+]o changes remained practically unaltered, and [K+]o changes were reduced by 20% on average. Selective degeneration of the pyramidal tract neurons was induced by performing chronic lesions of the pyramidal tract at the bulbar level. In these conditions, laminar profiles of amino acid-induced [Ca2+]o changes were considerably altered in the corresponding motor cortex. The maximum Ca2+ changes were then reduced by two-thirds, and the [Ca2+]o signals were practically constant throughout the cortex. Local cobalt (Co2+) or manganese (Mn2+) applications could abolish amino acid-evoked [Ca2+]o changes, whereas most of the [K+]o changes persisted. gamma-Aminobutyric acid applications could decrease [Ca2+]o signals to a large extent, whereas [K+]o changes were diminished by only 15-25% on average. These results show that the putative neurotransmitters Glu and Asp induce significant changes in [Ca2+]o and [K+]o in the rat cerebral cortex and indicate the possible origin of the ion fluxes in terms of neuronal elements and ionic channels: in particular, they indicate that the major part of the amino acid-induced [Ca2+]o maximum decreases can be ascribed to Ca2+ movements through channels located very likely on the pyramidal tract neurons in the motor cortex. They also indicate that significant reductions of the extracellular space volume occur during excitatory amino acid applications.

Amino Acids↗

The anticonvulsive action of adenosine: a postsynaptic, dendritic action by a possible endogenous anticonvulsant.

Neural afterdischarges generated in the presence of penicillin or low extracellular calcium concentrations were found to be inhibited by adenosine in the rat hippocampus in vitro. This anticonvulsant effect of adenosine is observed in the absence, as well as in the presence, of chemical synaptic transmission and apparently occurs at a postsynaptic site which is most sensitive in the apical dendritic region of the CA1 pyramidal cells. The methylxanthine theophylline antagonizes the effect of adenosine; and, the anticonvulsant action of the L-isomer of the adenosine analogue phenylisopropyladenosine (PIA) is substantially more potent than the D-isomer, findings which are characteristic of an A1 type adenosine receptor. The endogenous release of adenosine may therefore serve to tonically reduce the tendency for repetitive discharge in CA1 pyramidal cells via an interaction with a high affinity A1 receptor which appears to be preferentially localized in the apical dendrites.

Adenosine↗

GABA and baclofen reduce changes in extracellular free calcium in area CA1 of rat hippocampal slices.

Reductions in extracellular free Ca2+-concentration [( Ca2+]o) result from neuronal activation and can be induced by repetitive electrical stimulation or by application of excitatory amino acids. They reflect Ca2+-movements along its electrochemical gradient into pre- and postsynaptic elements. Amino acid-induced reductions in [Ca2+]o are predominantly caused by postsynaptic Ca2+ entry. Under conditions of blocked chemical synaptic transmission, a presynaptic component of extracellular Ca2+ loss becomes apparent during stimulation of the Schaffer collateral/commissural fibers system in stratum radiatum/moleculare. GABA, both iontophoretically and bath applied, always reduces pre- and postsynaptic components of [Ca2+]o changes. Baclofen regularly affects postsynaptic Ca2+ entry and has frequently also a suppressant action on presynaptic Ca2+ entry in area CA1.

Animals↗

Extracellular potassium concentration in chronic alumina cream foci of cats.

Changes in extracellular K+ concentration [( K+]o) were measured with ion-selective microelectrodes in chronic epileptic foci induced by topical application of A1(OH)3 cream on the sensorimotor cortex of cats. The foci were morphologically characterized by a scar surrounded by an area of marked gliosis. Base-line levels of [K+]o in gliotic tissue and its immediate border zone were comparable to those in normal cortical tissue. Peak levels of [K+]o obtained during repetitive electrical stimulation of the cortical surface and thalamic ventrobasal complex were only slightly enhanced with 11.6 mM in chronic foci and 10.8 mM in normal cortex. Iontophoretic K+ application into gliotic tissue was accompanied by slow negative potential shifts comparable to those observed in normal cortex. Passage of constant current through gliotic tissue caused local [K+]o changes in the vicinity of the current-passing electrode. Since these [K+]o changes were similar to those observed in normal tissue, it was concluded that the amount of transcellularly transported K ions was comparable in both tissues. Changes in the size of extracellular space (ES) were investigated by measuring local concentration changes of iontophoretically injected tetramethylammonium and choline ions. During stimulus-induced seizure activity, the ES shrank outside the gliotic area at sites of maximal [K+]o elevation, while it increased at sites within the gliotic tissue where [K+]o rises were smaller. The results suggest that the spatial buffer capacity of gliotic tissue for K+ is not severely impaired. Since the relationship between rises in [K+]o and subsequent undershoots at sites immediately bordering the gliotic tissue is comparable to that in normal cortex, the ability of this epileptic tissue for active K+ uptake appears to be unaffected. This conclusion is further supported by the observation that iontophoretically induced rises in [K+]o during undershoots are reduced to a similar extent as in normal cortex.

Aluminum Hydroxide↗

Presynaptic involvement in frequency facilitation in the hippocampal slice.

Orthodromic stimulation with frequencies from 1 to 30 Hz causes facilitation of extracellularly recorded population spikes and a decrease of extracellular Ca2+ concentration ( [Ca2+]o ) monitored with ion selective microelectrodes at the cell body layer of area CA1 of the hippocampal slice. Reducing the Ca2+ content of the perfusion medium impairs synaptic transmission. However, even under these conditions, weak stimulation evokes a significant decrease of [Ca2+]o, which is ascribed to a presynaptic Ca2+ entry. Stimulation with high intensities and/or frequencies initially induces an accelerated decrease in [Ca2+]o followed by the re-establishment of synaptic transmission, indicating a contribution of the presynaptic site to frequency facilitation.

Animals↗

Effects of GABA on presumed presynaptic Ca2+ entry in hippocampal slices.

Evoked field potentials and changes in [Ca2+]o were measured in the 'in vitro' hippocampal slice of the rat. When [Ca] in the perfusion medium was lowered to 0.2 mM synaptic transmission from Schaffer collateral/commissural fibers was blocked. Nevertheless, repetitive stimulation of afferent fibers still resulted in detectable decreases of [Ca2+]o. In contrast to findings in normal medium these decreases in [Ca2+]o could be larger in stratum radiatum than in stratum pyramidale, so mimicking the spatial distribution of activated afferent fibers. These findings suggest, that the loss of extracellular Ca2+ in low Ca2+ media is predominantly due to entry into presynaptic terminals. This permits to study effects of drugs on presynaptic endings. We found that iontophoretic application of GABA is capable to block this presumed presynaptic Ca2+ entry without affecting the electrical activity of the afferent fibers. This suggests, that presynaptic GABA receptors occur also in the Schaffer collateral/commissural fiber system.

Animals↗

Aspartate and glutamate induced reductions in extracellular free calcium and sodium concentration in area CA1 of 'in vitro' hippocampal slices of rats.

Extracellular Ca2+ and Na+ were simultaneously measured with ion selective electrodes in the CA1 pyramidal cell layer of rat hippocampal slices. Repetitive stimulation of the Schaffer collateral/commissural fiber system led to decreases in [Ca2+] omicron and [Na+] omicron (delta Ca, delta Na) by up to 0.5 mM and 13 mM, respectively. Iontophoretic application of glutamate or aspartate caused dose-dependent delta Ca of more than 1 mM and delta Na of more than 45 mM. Such ionic alterations were smaller in the presence of GABA or of the Ca2+ antagonists Ni2+ and Mg2+. Ca2+ signals were diminished when evoked in low-Na+ bathing medium and Na+ signals were reduced in Ca2+-free solutions. Thus glutamate- and aspartate-dependent decreases in [Na+] omicron and [Ca2+] omicron appear to be caused by 3 different mechanisms: Na+ movement through amino acid-activated ionophores; Ca2+ movement through selective channels, and Ca2+-dependent Na+ movement.

Animals↗

Spontaneous epileptiform activity of CA1 hippocampal neurons in low extracellular calcium solutions.

Lowering extracellular [Ca2+] in rat hippocampal slices induces spontaneous epileptiform activity in area CA1, which is characterized by rhythmic burst firing of CA1 neurons and by prolonged negative potential shifts at the pyramidal cell body layer. This activity is accompanied by transient decreases of [Na+] and increases of [K+] in the extracellular space. In spite of the complete blockade of synaptic transmission, the wave of epileptiform activity propagates across area CA1. These findings suggest, that non-synaptic mechanisms may play a role in the generation and spread of epileptiform activity in the mammalian CNS.

Animals↗

Changes in [Ca2+]o and [K+]o during repetitive electrical stimulation and during pentetrazol induced seizure activity in the sensorimotor cortex of cats.

Changes in [Ca2+]o and [K+]o were measured in the sensorimotor cortex of cats during repetitive electrical stimulation and during pentetrazol induced epileptiform activity. Repetitive stimulation of the thalamic ventrobasal complex (VB) or of the cortical surface (CS) caused decreases in [Ca2+]o by up to 0.45 mM and increases in [K+]o by up to 7 mM. Maximum reductions of [Ca2+]o delta [Ca2+]o were found in depths of 100 to 300 micrometers below cortical surface, while rises in [K+]o were largest in depths of 600 to 1000 micrometers dependent on stimulation site. At depths below 700-900 micrometers increases in [K+]o were often accompanied by rises in [Ca2+]o of about 0.2 mM. Pentetrazol (PTZ) when injected at doses of 25 to 40 mg/kg body weight induced spontaneous seizure activity, which was in about 40% preceded by a slight fall of baseline [Ca2+]o. Repetitive stimulation and spontaneous seizures resulted in delta [Ca2+]o of up to 0.6 mM, whereas rises in [K+]o remained limited to a 'ceiling level' of about 10 mM. After PTZ application, peak delta [Ca2+]o were found at the same recording sites, but, in contrast to normal cortex, decreases in [Ca2+]o were observed in all cortical layers. The enhanced Ca2+-signals after PTZ application and the observed reduction of [Ca2+]o before seizure onset suggest that PTZ utilizes Ca2+-dependent mechanisms to initiate seizure activity.

Animals↗

Changes in [Ca2+]0, [K+]0 and neuronal activity during oenanthotoxin-induced epilepsy in cat sensorimotor cortex.

Effects of the convulsant drug oenanthotoxin on field potentials, neuronal discharges and extracellular free Ca2+ and K+ concentrations were studied in the sensorimotor cortex of cats. Fifteen to 20 min after topical OETX application spike activity developed (surface negative, deeper layers positive) with a frequency of 0.5-1/sec. With the maturation of the focus spikes became multipeaked and the reversal point of field potential polarity shifted deeper into the cortex. Spontaneous seizures developed when the spike activity had become negative in polarity in recording depths of 1300 micron below the cortical surface. Spontaneous seizures were associated with large rises in [K+]0 and reductions in [Ca2+]0 (delta [K+]0, delta [Ca]0). The latter were often larger than maximum changes in normal cortex. The decay in [Ca2+]0 associated with spontaneous seizures tended to precede the onset of ictal activity. The findings suggest that OETX, like other epileptogenic drugs, uses Ca2+-dependent mechanisms for the initiation of epileptiform activity.

Alkynes↗

Crystallographic study of mechanism of ribonuclease T1-catalysed specific RNA hydrolysis.

Ribonuclease T1 (RNase T1) cleaves the phosphodiester bond of RNA specifically at the 3'-end of guanosine. 2'-guanosinemonophosphate (2'-GMP) acts as inhibitor for this reaction and was cocrystallized with RNase T1. X-Ray analysis provided insight in the geometry of the active site and in the parts of the enzyme involved in the recognition of guanosine. RNase T1 is globular in shape and consists of a 4.5 turns alpha-helix lying "below" a four-stranded antiparallel beta-sheet containing recognition center as well as active site. The latter is indicated by the position of phosphate and sugar residues of 2'-GMP and shows that Glu58, His92 and Arg77 are active in phosphodiester hydrolysis. Guanine is recognized by a stretch of protein from Tyr42 to Tyr45. Residues involved in recognition are peptide NH and C = O, guanine O6 and N1H which form hydrogen bonds and a stacking interaction of Tyr45 on guanine. Although, on a theoretical basis, many specific amino acid-guanine interactions are possible, none is employed in the RNase T1.guanine recognition.

Amino Acid Sequence↗

Crystal and molecular structure of the sulfhydryl protease calotropin DI at 3.2 A resolution.

The three-dimensional structure of the sulfhydryl protease calotropin DI from the madar plant, Calotropis gigantea, has been determined at 3.2 A resolution using the multiple isomorphous replacement method with five heavy atom derivatives. A Fourier synthesis based on protein phases with a mean figure of merit of 0.857 was used for model building. The polypeptide backbone of calotropin DI is folded to form two distinct lobes, one of which is comprised mainly of alpha-helices, while the other is characterized by a system of all antiparallel pleated sheets. The overall molecular architecture closely resembles those found in the sulfhydryl proteases papain and actinidin. Despite the unknown amino acid sequence of calotropin DI a number of residues around its active center could be identified. These amino acid side-chains were found in a similar arrangement as the corresponding ones in papain and actinidin. The polypeptide chain between residues 1 and 18 of calotropin DI folds in a unique manner, providing a possible explanation for the unusual inability of calotropin DI to hydrolyze those synthetic substrates that papain and actinidin act upon.

Binding Sites↗

Specific protein-nucleic acid recognition in ribonuclease T1-2'-guanylic acid complex: an X-ray study.

RNase T1 is folded into an alpha-helix of 4.5 turns, covered by a four-strand antiparallel beta-sheet. Specific recognition of 2'-guanylic acid arises from hydrogen bonding between main chain peptide groups and the O-6 and N-1-H of guanine, as well as from stacking of Tyr 45 on guanine. At the active site, Glu 58, His 92 and Arg 77 are involved in phosphodiester hydrolysis.

Amino Acid Sequence↗

Aspartate-induced changes in extracellular free calcium in 'in vitro' hippocampal slices of rats.

Extracellular free calcium ([Ca2+]0) and neuronal activity were measured with double-barreled Ca2+-selective/reference microelectrodes in the CA1 field of rat hippocampal slices during stimulation of Schaffer collaterals and during iontophoretic application of the excitatory amino acid aspartate (Asp). Baseline [Ca2+]0 was 1.5 mM. Repetitive stimulation and aspartate application caused decreases in [Ca2+]0 (delta Ca), which were largest in the pyramidal cell layer. During iontophoretic application of GABA, aspartate often failed to induce significant alterations in [Ca2+]0. Mg2+ (9 mM) and Ni2+ (3mM) when added to the perfusion medium reduced aspartate-dependent delta Ca by 15 and 70% respectively. In tetrodotoxin (10(-6) M) containing Ringer the aspartate-induced delta Ca were about 10% smaller. These findings suggest that the decreases in [Ca2+]0 are mainly due to a postsynaptic Ca2+ entry through selective Ca2+ conductances.

Animals↗

Stimulus-induced changes in extracellular Na+ and Cl- concentration in relation to changes in the size of the extracellular space.

Extracellular Na+ - and Cl- -concentrations ([Na+]o, [Cl-]o) were recorded with ion-selective microelectrodes during repetitive stimulation and stimulus-induced self-sustained neuronal afterdischarges (SAD) in the sensorimotor cortex of cats. In all cortical layers [Na+]o initially decreased by 4-7 mM. In depths of more than 600 micrometer below the cortical surface such decreases usually turned into increases of 2-6 mM during the course of the SADs, whereas in superficial layers [Na+]o never rose above its resting level. [Cl-]o always showed an increase in the course of the SADs often preceded by an initial small decrease. The average increase at a depth of 1,000 micrometers was about 7 mM. [Cl-]o reached peak values at about the end of the ictal period, whereas [Na+]o reached its maximum shortly after the end of the SAD, at times when [K+]o was still elevated above the baseline concentration. These data indicate that the extracellular osmolarity can increase during SAD by up to 30 mM. Such an increase in osmolarity can be explained by an increase in the number of intracellular particles, caused by cleavage of larger molecules during enhanced metabolism. This could lead to cell-swelling due to passive water influx from the extracellular space (ES). However, the resulting reduction of the size of the ES is calculated to be less than 10% for an increase in intracellular osmolarity by 30 mOsm. This value is too small as compared to previously measured ES-reductions under similar conditions (i.e., 30% reduction at 1,000 micrometers; Dietzel et al. 1980). Reductions of the size of the ES that accompany the observed changes in the ionic environment, are quantitatively explained on the basis of the extended glial buffering mechanism described in the preceding paper.

Animals↗