Broken-dimer model in a-Si:H.
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Journal Article↗
Biomedical subjects
D Muller
Publications and source records attributed to D Muller.
Evidence that changes in presynaptic calcium currents are not responsible for long-term potentiation in hippocampus.
We used two approaches to test the possibility that changes in presynaptic calcium currents might be responsible for the long-term potentiation (LTP) effect induced by high-frequency stimulation in area CA1 of hippocampal slices. In a first series of experiments, we compared the effect of LTP induction on paired-pulse facilitation with that produced by changes in extracellular calcium concentration, a procedure that modifies presynaptic calcium currents during depolarization by changing the ionic gradient for calcium. In hippocampus, as in peripheral synapses, increasing concentrations of extracellular calcium caused a marked reduction in the degree of facilitation obtained with paired-pulse stimulation; LTP, conversely, did not affect the facilitation ratio. The differential effect of changing calcium concentrations versus LTP induction on paired-pulse facilitation was observed with different interpulse intervals as well as in conditions in which the changes in response size produced by the two manipulations were comparable. In the second approach, we measured calcium dependency curves of synaptic responses before and after LTP induction or application of 4-aminopyridine, a blocker of potassium channels that increases presynaptic calcium currents by slowing spike repolarization. Procedures that increase calcium entry into terminals during transmission should shift to the left the sigmoidal function relating extracellular calcium to the slope of the extracellular response. This in turn should result in disproportionate effects of the procedure as a function of the calcium concentration. This prediction was realized with 4-aminopyridine but did not occur following LTP induction: control and potentiated responses were similarly affected by changes in calcium concentration. Although indirectly, these data strongly suggest that LTP is not accompanied by alterations in the presynaptic calcium dynamics associated with transmitter release.
Action Potentials↗
The NMDA receptor-mediated components of responses evoked by patterned stimulation are not increased by long-term potentiation.
The participation of N-methyl-D-aspartate (NMDA) receptors in synaptic transmission before and after induction of long-term potentiation (LTP) was studied in field CA1 of hippocampal slices. NMDA receptor-mediated postsynaptic responses were determined by comparing responses recorded in the presence and absence of the selective antagonist, D-2-amino-5-phosphonopentanoate (D-AP5, 50 microM). In the presence of physiological magnesium concentrations (1 mM), robust D-AP5-sensitive responses could be evoked by high frequency bursts (4 pulses, 100 Hz) when burst stimulation was preceded 200 ms earlier by 'priming' stimulation (2 pulses, 15 ms apart) of a separate input. Induction of LTP resulted in a substantial potentiation (35%) of non-NMDA-mediated responses to primed bursts but not of NMDA-mediated responses. These results suggest that long-term postsynaptic modifications are at least partly responsible for the expression of LTP.
Animals↗
Long-lasting physiological effects of bath applied N-methyl-D-aspartate.
The present experiments describe a long-lasting form of potentiation induced in field CA1 of rat hippocampal slices by bath application of N-methyl-D-aspartate (NMDA), in association with low magnesium concentrations, glycine and spermine. The potentiation effect consisted of a 50% increase in slope of field potentials and was stable for at least 80 min post treatment. It was not accompanied by detectable changes in antidromic responses and was completely blocked by an antagonist of NMDA receptor. The possible relationship of pharmacologically induced potentiation to long-term potentiation (LTP) is discussed.
2-Amino-5-phosphonovalerate↗
Rate-limiting step for transmission at excitatory synapses in hippocampus.
To examine mechanisms that might be responsible for limiting transmission at excitatory synapses in hippocampus, we analyzed the relationship between extracellular calcium concentrations (1-6 mM) and postsynaptic responses in field CA1 of hippocampal slices using low stimulation intensities and a paired-pulse paradigm. Three effects were observed: One, the relationship between calcium levels and the slope (or amplitude) of the postsynaptic response was described by a sigmoidal function with an asymptote at about 4 mM. Double reciprocal pilots relating calcium concentration to the initial slope of EPSPs provided evidence for the cooperativity expected between calcium ions and transmitter release. Two, both the rise time and half-decay time of the postsynaptic responses were reduced with increasing calcium concentrations. These effects of calcium were more pronounced on the first response elicited by paired-pulse stimulation and were considerably attenuated by 2 microM bicuculline, indicating that feed-forward inhibition was positively related to calcium concentration and differentially activated by repetitive stimulation. However, inhibition was not responsible for the asymptotic relationship observed between calcium and response size. Three, while increasing the calcium concentration beyond 4 mM did not further affect the initial slope of excitatory postsynaptic potentials (EPSPs), paired-pulse facilitation and 4-aminopyridine were still effective in increasing response size. These results suggest 1) that neither the number of postsynaptic receptors nor the number of transmitter quanta available for release were limiting transmission as a function of the calcium concentration; and 2) that calcium entry into presynaptic terminals was likely to represent the limiting step under the conditions used.
4-Aminopyridine↗
Momentary alteration of the postsynaptic membrane during transmission of a single nerve impulse.
Transmission of a nerve impulse at neuromuscular and other synapses is an extremely brief event. By using rapid-freezing and cryofracture techniques in the electric organ of Torpedo, synaptic transmission was found to be accompanied by significant changes affecting the postsynaptic membrane for a few milliseconds. In the replicas, the protoplasmic leaflet of this membrane was seen to contain intramembrane particles (IMPs) of two different forms, globular and elongated. Globular IMPs had a mean diameter of 8.8 nm; they were the most frequently found (80% in unstimulated specimens). Elongated IMPs had a major diameter of 17.9 nm, about twice that of globular IMPs. Transmission of a single nerve impulse was accompanied by a marked decrease in the number of globular IMPs and by an increase in the number of elongated IMPs, as if there were a coalescence of two adjacent round particles to form an elongated one. These changes started soon after the electrical stimulus and lasted for approximately 3 ms. IMPs in the postsynaptic protoplasmic face are thought to correspond to a certain proportion of nicotinic acetylcholine receptors that were extracted with this leaflet during the fracture process. The phenomenon described here reflects an abrupt change in the membrane, probably linked to activation of the acetylcholine nicotinic receptors.
Animals↗
Contributions of quisqualate and NMDA receptors to the induction and expression of LTP.
The contributions of two subclasses of excitatory amino acid transmitter receptors to the induction and expression of long-term potentiation (LTP) were analyzed in hippocampal slices. The quisqualate/kainate receptor antagonist DNQX (6,7-dinitro-quinoxaline-2,3-dione) blocked 85% of the evoked field potential, leaving a small response that was sensitive to D-AP5 (D-2-amino-5-phosphonopentanoate), an N-methyl-D-aspartate (NMDA) receptor blocker. This residual D-AP5-sensitive response was of comparable size in control and previously potentiated inputs. High-frequency stimulation in the presence of DNQX did not result in the development of robust LTP. Washout of the drug, however, revealed the potentiation effect. Thus NMDA-mediated responses can induce, but are not greatly affected by, LTP; non-NMDA receptors, conversely, mediate responses that are not needed to elicit LTP but that are required for its expression.
2-Amino-5-phosphonovalerate↗
Random oligonucleotide mutagenesis: application to a large protein coding sequence of a major histocompatibility complex class I gene, H-2DP.
We have used random oligonucleotide mutagenesis (or saturation mutagenesis) to create a library of point mutations in the alpha 1 protein domain of a Major Histocompatibility Complex (MHC) molecule. This protein domain is critical for T cell and B cell recognition. We altered the MHC class I H-2DP gene sequence such that synthetic mutant alpha 1 exons (270 bp of coding sequence), which contain mutations identified by sequence analysis, can replace the wild type alpha 1 exon. The synthetic exons were constructed from twelve overlapping oligonucleotides which contained an average of 1.3 random point mutations per intact exon. DNA sequence analysis of mutant alpha 1 exons has shown a point mutant distribution that fits a Poisson distribution, and thus emphasizes the utility of this mutagenesis technique to "scan" a large protein sequence for important mutations. We report our use of saturation mutagenesis to scan an entire exon of the H-2DP gene, a cassette strategy to replace the wild type alpha 1 exon with individual mutant alpha 1 exons, and analysis of mutant molecules expressed on the surface of transfected mouse L cells.
Amino Acid Sequence↗
Ultrasound angioscopy: real-time, two-dimensional, intraluminal ultrasound imaging of blood vessels.
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Animals↗
The collagenase gene family in humans consists of at least four members.
Stromelysin is a collagenase-related connective-tissue-degrading metalloproteinase. We have detected RNAs capable of hybridizing to a rat stromelysin cDNA in 11 of 69 human tumours tested. Molecular cloning of cDNAs to these RNAs has identified them as a mixture of stromelysin RNA and a transcript of a hitherto-undescribed related human gene, the stromelysin-2 gene. We have also isolated cDNAs corresponding to a more distantly related new human gene, the pump-1 gene. A comparison of the cDNA-derived amino acid sequences of stromelysin-2 and pump-1 with the known sequences of stromelysin and collagenase reveals significant similarities, with conservation of sequence motifs believed to have functional importance in metalloproteinase action. We conclude that the collagenase gene family in humans consists of at least four members, and speculate that expression of these genes plays a role in cancer.
Amino Acid Sequence↗
Dextran-coated silica packings for high-performance size-exclusion chromatography of proteins.
Porous silica beads have excellent mechanical properties for the high-performance liquid chromatography of proteins. However, the importance of non-specific interactions between silanols on the silica surface and the proteins requires a modification of these supports before they can be used as stationary phases for size-exclusion chromatography (SEC). Silica beads were coated with dextran bearing a small number of positive charges in order to neutralize the negatively charged silanol groups. Diethylaminoethyl-dextrans (DEAE-dextrans) with a relatively low percentage of dextran units bearing DEAE functions were layered on the silica beads. The influence of several characteristics of these packings on chromatographic performance were studied in order to determine the optimal conditions for the SEC of proteins.
Chromatography, DEAE-Cellulose↗
N-methyl-D-aspartate receptor-mediated component of synaptic responses to single-pulse stimulation in rat hippocampal slices.
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2-Amino-5-phosphonovalerate↗
Long-term potentiation: persisting problems and recent results.
In this paper we discuss recent experimental results pertinent to three unresolved issues regarding the long-term potentiation (LTP) effect: the nature of its enduring substrates, the biochemical mechanisms that produce it, and its potential role in memory. LTP appears to be triggered by a postsynaptic influx of calcium and is associated with alterations in the shape of dendritic spines and probably the formation of new synapses. We discuss the possibility that morphological reorganization also modifies membrane surface chemistry of synaptic elements. Evidence is presented that LTP is not associated with changes in presynaptic calcium currents. Activation of protein kinase C is shown to be insufficient for the induction of LTP, although it may play a modulatory role. The hypothesis that activation of a calcium-sensitive protease (calpain) is pivotal to the establishment of LTP is supported by experiments showing that a calpain inhibitor, leupeptin, blocks LTP. Furthermore, activation of NMDA receptors, an event implicated in LTP induction, is accompanied by calcium-sensitive proteolysis of spectrin, a major dendritic cytoskeletal protein. The finding that stimulation patterns designed to mimic naturally-occurring cell discharge patterns are highly effective for LTP induction greatly strengthens the hypothesis that LTP actually occurs during the encoding of information in cortical systems. Potential contributions of LTP to learning are explored using computer simulations of a simple cortical network.
Animals↗
Phorbol ester-induced synaptic facilitation is different than long-term potentiation.
The studies described here tested the hypothesis that the changes in synaptic efficacy produced by phorbol esters in hippocampal slices are equivalent to the long-term potentiation (LTP) induced by high-frequency stimulation. In contrast to the extremely stable synaptic potentiation induced by electrical stimulation, the facilitatory effects of phorbol 12,13-diacetate and phorbol 12,13-dibutyrate were transient: washout of the drugs restored normal responses in approximately 1-2 and 2-4 hr for phorbol diacetate and phorbol dibutyrate, respectively. It is noteworthy that the more liposoluble of the phorbol esters required longer washout periods. Robust LTP still occurred in response to high-frequency stimulation after washout of phorbol esters and to a lesser degree during their application. Treatment of slices with H-7, an inhibitor of protein kinase C, did not prevent LTP induction although it significantly affected neuronal excitability and produced effects opposite to those of phorbol esters. Finally, phorbol esters altered responses to repetitive stimulation in a way that could account for the reduced LTP elicited in their presence. These results indicate that the increases in synaptic responses caused by phorbol esters and high-frequency electrical stimulation are quite different and thus do not support the hypothesis that activation of protein kinase C, the presumed target of the phorbol esters, triggers LTP.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Long-term potentiation differentially affects two components of synaptic responses in hippocampus.
We have used low magnesium concentrations and the specific antagonist D-2-amino-5-phosphonopentanoate (D-AP5) to estimate the effects of long-term potentiation (LTP) on the N-methyl-D-aspartate (NMDA) and non-NMDA receptor-mediated components of postsynaptic responses. LTP induction resulted in a considerably larger potentiation of non-NMDA as opposed to NMDA receptor-related currents. Increasing the size of postsynaptic potentials with greater stimulation currents or with paired-pulse facilitation produced opposite effects; i.e., those aspects of the response dependent on NMDA receptors increased to a greater degree than did those components mediated by non-NMDA receptors. These results pose new constraints on hypotheses about the locus and nature of LTP and strongly suggest that postsynaptic modifications are part of the effect.
2-Amino-5-phosphonovalerate↗
Energy metabolism and quantal acetylcholine release: effects of botulinum toxin, 1-fluoro-2,4-dinitrobenzene, and diamide in the Torpedo electric organ.
In the Torpedo electric organ, a modified nerve-muscle system, type A botulinum toxin blocked the release of acetylcholine (ACh) quanta, both neurally evoked and spontaneous. At the same time, the toxin increased the release of a class of small miniature potentials (the subminiature potentials), reduced the ATP and more the creatine phosphate content of the tissue, and impaired the activity of creatine kinase (CK). Thus, we compared this pattern of changes with those provoked by 1-fluoro-2,4-dinitrobenzene (FDNB), an efficient inhibitor of CK. As expected, FDNB rapidly inactivated CK, which resulted in a profound depletion of ATP whereas the stores of creatine phosphate were preserved. In addition, FDNB caused conspicuous morphological alterations of nerve endings and ACh depletion. This agent also suppressed evoked and spontaneous quantal release whereas the occurrence of subminature potentials was markedly increased. Diamide, a penetrating thiol oxidizing substance, provoked first a transient rise in quantal ACh release and then blockade of transmission with, again, production of a large number of subminiature potentials. Creatine phosphate was depleted in the tissue by diamide, the ATP content reduced, and CK activity partly inhibited. The morphology of nerve terminals did not show obvious changes with either diamide or botulinum toxin at the stage of transmission failure. Although the three poisons acted by different mechanisms, this resulted in a rather similar pattern of physiological changes: failure of quantal release and enhancement of subquantal release. These results and experiments on synaptosomes indicated that CK inhibition was probably a crucial mechanism for FDNB but not for diamide or botulinum intoxication.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetylcholine↗
Functional reconstitution of N-methyl-D-aspartate receptors in artificial lipid bilayers.
Functional reconstitution of the N-methyl-D-aspartate (NMDA) receptors was achieved by adding synaptic membranes from rat brain to large planar bimolecular lipid membranes (BLMs). The reconstituted receptors exhibited several properties of the NDMA receptors described using a variety of biochemical and electrophysiological techniques. Addition of NMDA at concentrations between 5 and 50 microM produced large, voltage-dependent increases in membrane conductivity. The selective antagonist of NMDA receptors, amino-2-phosphonopentanoate (AP-7), totally blocked the response of the bilayers to NMDA as did micromolar concentrations of magnesium; this latter effect was also voltage-dependent. These results indicate that BLMs can be used to study the ion channels and regulatory processes associated with NMDA receptors from adult brain in ways that could not be accomplished with conventional neurophysiological techniques.
Aspartic Acid↗
Adhesion of human breast cancer cell line MCF-7 to human vascular endothelial cells in culture. Enhancement by activated platelets.
The interactions of MCF-7 tumor cells with human vascular endothelial cells (EC) and subendothelial extracellular matrices (ECM) were morphologically observed by electron microscopy and quantitatively evaluated by labelling tumor cells with 111Indium-oxine. MCF-7 tumor cells adhered more rapidly to ECM than to the apical surface of a confluent monolayer of EC. The affinity of MCF-7 cells for type-IV collagen was greater than for fibronectin, suggesting that type-IV collagen contributes to the higher rate of adhesion of MCF-7 cells to the subendothelial ECM. Otherwise, the attachment of tumor cells to EC was increased in the presence of both washed platelets and 0.1% citrated platelet-poor plasma (cPPP), a condition accelerating platelet aggregation by tumor cells. The enhancement of MCF-7 adhesion to EC in the presence of platelets and cPPP was completely blocked by the addition of prostacyclin, or hirudin, a specific thrombin inhibitor. In ultrastructural studies, MCF-7 initiated EC retraction, and firm attachment and flattening occurred on exposed ECM. When MCF-7 cells were incubated with platelets and cPPP, most of the tumor cells adhering to the EC and inducing disruption of endothelial monolayer were closely packed and associated with platelet aggregates. MCF-7 cells appeared to adhere more efficiently to exposed subendothelial ECM when they were associated into multicellular aggregates containing platelets and trapped in a fibrin thrombus. Thus, this homologous human system of cultured vascular EC and breast carcinoma line MCF-7 cells may be used to assess anti-aggregant compounds for their ability to alter tumor-cell implantation on EC-lined surfaces.
Adenosine Diphosphate↗