Microarray analysis of differentially expressed genes after exposure of normal human fibroblasts to ionizing radiation from an external source and from DNA-incorporated iodine-125 radionuclide.

Exposure of cells to ionizing radiation (IR) produces changes in the expression level of a large number of genes. However, less is known of gene-expression changes caused by local radiation exposure from radionuclides within cells. We studied changes in the genome-wide gene expression induced by decay of 125I incorporated into DNA as [125I]-iododeoxyuridine (125I-IUdR) in normal IMR-90 human lung fibroblasts and compared them with the changes produced by external gamma-radiation delivered at high (HDR) or low (LDR) dose rate. We found that more than 2000 genes were consistently up- or down-regulated following HDR and LDR gamma-radiation. The profiles of differentially expressed genes following HDR and LDR shared about 64% (up) and 74% (down) genes in common, with many genes identified as radiation-responsive for the first time. In contrast, in all only 206 genes changed their expression level in the 125I-IUdR-treated cells, even though the total number of DNA double-strand breaks (DSB) produced by 125I-IUdR exceeded that produced by the gamma-radiation. With few exceptions, the expression levels of 125I-IUdR-responsive genes were also altered following gamma-irradiation. Therefore, nuclear DNA-localized decays of 125I produce 10 times fewer differentially expressed genes than whole-cell exposure to gamma-radiation of comparable dose. These results suggest that the effect of IR on the changes in global gene expression depends on the distribution of energy depositions within the cell. In contrast to cell survival, DNA DSB may not be the major factor modulating changes in gene expression following irradiation.

Quantal analysis suggests strong involvement of presynaptic mechanisms during the initial 3 h maintenance of long-term potentiation in rat hippocampal CA1 area in vitro.

Long-term potentiation (LTP) is the most prominent model to study neuronal plasticity. Previous studies using quantal analysis of an early stage of LTP in the CA1 hippocampal region (<1 h after induction) suggested increases in both the mean number of transmitter quanta released by each presynaptic pulse (m, quantal content) and postsynaptic effect of a single quantum (v, quantal size). When LTP was large, it was m that increased predominantly suggesting prevailing presynaptic contribution. However, LTP consists of several temporary phases with presumably different mechanisms. Here we recorded excitatory postsynaptic potentials from CA1 hippocampal slices before and up to 3.5 h after LTP induction. A new version of the noise deconvolution revealed significant increases in m with smaller and often not statistically significant changes in v. The changes in m were similar for both early (<1 h) and later (1-3 h) post-tetanic periods and correlated with LTP magnitude. The coefficient of variation of the response amplitude and the number of failures decreased during both early and late post-tetanic periods. The results suggest that both early (<0.5 h) and later LTP components (0.5-3 h) are maintained by presynaptic changes, which include increases in release probabilities and the number of effective release sites. In addition initially silent synapses can be converted into effective ones due to either pre- or postsynaptic rearrangements. If this occurs, our data indicate that the number and the efficacy of the receptors in the new transmission sites are approximately similar to those in the previously effective sites.

Differences in amplitude-voltage relations between minimal and composite mossy fibre responses of rat CA3 hippocampal neurons support the existence of intrasynaptic ephaptic feedback in large synapses.

Computer simulations and electrophysiological experiments have been performed to test the hypothesis on the existence of an ephaptic interaction in purely chemical synapses. According to this hypothesis, the excitatory postsynaptic current would depolarize the presynaptic release site and further increase transmitter release, thus creating an intrasynaptic positive feedback. For synapses with the ephaptic feedback, computer simulations predicted non-linear amplitude-voltage relations and voltage dependence of paired-pulse facilitation. The deviation from linearity depended on the strength of the feedback determined by the value of the synaptic cleft resistance. The simulations showed that, in the presence of the intrasynaptic feedback, recruitment of imperfectly clamped synapses and synapses with linear amplitude-voltage relations tended to reduce the non-linearity and voltage dependence of paired-pulse facilitation. Therefore, the simulations predicted that the intrasynaptic feedback would particularly affect small excitatory postsynaptic currents induced by activation of electrotonically close synapses with long synaptic clefts. In electrophysiological experiments performed on hippocampal slices, the whole-cell configuration of the patch-clamp technique was used to record excitatory postsynaptic currents evoked in CA3 pyramidal cells by activation of large mossy fibre synapses. In accordance with the simulation results, minimal excitatory postsynaptic currents exhibited "supralinear" amplitude-voltage relations at hyperpolarized membrane potentials, decreases in the failure rate and voltage-dependent paired-pulse facilitation. Composite excitatory postsynaptic currents evoked by activation of a large amount of presynaptic fibres typically bear linear amplitude-voltage relationships and voltage-independent paired-pulse facilitation. These data are consistent with the hypothesis on a strong ephaptic feedback in large mossy fibre synapses. The feedback would provide a mechanism whereby signals from large synapses would be amplified. The ephaptic feedback would be more effective on synapses activated in isolation or together with electrotonically remote inputs. During synchronous activation of a large number of neighbouring inputs, suppression of the positive intrasynaptic feedback would prevent abnormal boosting of potent signals.

Postsynaptic hyperpolarization increases the strength of AMPA-mediated synaptic transmission at large synapses between mossy fibers and CA3 pyramidal cells.

In chemical synapses information flow is polarized. However, the postsynaptic cells can affect transmitter release via retrograde chemical signaling. Here we explored the hypothesis that, in large synapses, having large synaptic cleft resistance, transmitter release can be enhanced by electrical (ephaptic) signaling due to depolarization of the presynaptic release site induced by the excitatory postsynaptic current itself. The hypothesis predicts that, in such synapses, postsynaptic hyperpolarization would increase response amplitudes "supralinearly", i.e. stronger than predicted from the driving force shift. We found supralinear increases in the amplitude of minimal excitatory postsynaptic potential (EPSP) during hyperpolarization of CA3 pyramidal neurons. Failure rate, paired-pulse facilitation, coefficient of variation of the EPSP amplitude and EPSP quantal content were also modified. The effects were especially strong on mossy fiber EPSPs (MF-EPSPs) mediated by the activation of large synapses and identified pharmacologically or by their kinetics. The effects were weaker on commissural fiber EPSPs mediated by smaller and more remote synapses. Even spontaneous membrane potential fluctuations were associated with supralinear MF-EPSP increases and failure rate reduction. The results suggest the existence of a novel mechanism for retrograde control of synaptic efficacy from postsynaptic membrane potential and are consistent with the ephaptic feedback hypothesis.

Intracellular studies of the interaction between paired-pulse facilitation and the delayed phase of long-term potentiation in the hippocampus.

The mechanisms of the early (up to 1 h) and late (up to 3 h) phases of long-term potentiation were studied by analyzing the interaction between long-term potentiation and presynaptic paired-pulse facilitation. "Minimal" excitatory postsynaptic potentials were measured in pyramidal neurons in field CA1 of rat hippocampal slices in conditions of paired-pulse stimulation of the radial layer. In most neurons, paired-pulse facilitation decreased after induction of long-term potentiation, and this reduction lasted throughout the recording period (up to 3.5 h). Changes in paired-pulse facilitation correlated with the extent of long-term facilitation and with the initial level of paired-pulse facilitation, and the extent of facilitation depended on the initial level of paired-pulse facilitation. This latter relationship was different for the early and late phases of long-term potentiation and increased with time. Overall, the data obtained here demonstrate a significant role for presynaptic mechanisms in maintaining both the early and late phases of long-term potentiation. It is suggested that the basic mechanism of the early phase of potentiation is an increase in the probability that transmitter will be released, which also leads to an increase in the number of effective release sites, due to transformation of "presynaptically quiet" synapses into effective synapses. It is proposed that the development of the late phase is based on simultaneous pre- and postsynaptic structural transformations which increase the number of synaptically active zones.

[Intracellular study of interaction of paired-pulse facilitation and late phase of hippocampal long-term potentiation]. / Vnutrikletochnoe issledovanie vzaimodeistviia mezhdu parnoi fasilitatsiei i pozdnei fazoi dolgovremennoi gippokampal'noi potentsiatsii.

Presynaptic paired-pulse facilitation (PPF) rate decreased in most CA1 pyramidal neurones following the long-term potantiation (LTP) induction. The decrease correlated with the LTP magnitude as well as with the initial (pretetanic) PPF rate. The data obtained suggests an involvement of presynaptic mechanisms in maintaining the early and the delayed LTPs.

Interaction between paired-pulse facilitation and long-term potentiation of minimal excitatory postsynaptic potentials in rat hippocampal slices: a patch-clamp study.

Long-term potentiation is an experimental paradigm used to study synaptic plasticity and memory mechanisms. One similarity between long-term potentiation and memory is the existence of several distinct phases. However, our preliminary quantal analysis did not reveal essential differences in expression mechanisms of the early (< 1 h) and later (up to 3 h) phases of long-term potentiation. The data were compatible with presynaptic mechanisms of both phases. Another approach to distinguish between presynaptic and postsynaptic mechanisms is analysis of interaction between long-term potentiation and presynaptic paired-pulse facilitation. Such analysis had been previously done mainly with recordings of field potentials reflecting the activity of large neuronal populations. Only the early potentiation phase had been previously analysed with recordings from single neurons. The results from different groups were contradictory. In the present study, minimal excitatory postsynaptic potentials were recorded from CA1 pyramidal neurons of rat hippocampal slices. Paired-pulse facilitation ratios were calculated for various periods (up to 2-3 h) following induction of long-term potentiation. The ratio persistently decreased in the majority of neurons following long-term potentiation induction. The decrease in the paired-pulse facilitation ratio correlated with the magnitude of long-term potentiation and with the initial (pretetanic) facilitation ratio. Therefore, the general results of the present analysis was similar with the results of the quantal analysis: it is consistent with a strong involvement of presynaptic mechanisms in maintenance of both early and late phases of long-term potentiation. However, individual neurons could show variable changes in the paired-pulse facilitation, e.g., increases at late (> 0.5-1 h) periods after tetanus. Calculations of partial correlations and regression analysis indicated that positive correlation between potentiation magnitude and initial (pretetanic) paired-pulse facilitation tended to increase in the late potentiation phase (1.5-2.5 h post-tetanus) indicating that different mechanisms are involved in the early (0.5 h post-tetanus) and the late phase of long-term potentiation. The findings are compatible with involvement of presynaptic mechanisms in both the early and late phases of long-term potentiation. However, the results suggest that contribution of changes in release probability and in effective number of transmitter release sites may differ during the two phases. It is suggested that activation of silent synapses and increases in the number of transmission zones due to pre- and postsynaptic structural rearrangements represent important mechanisms of the late phase of long-term potentiation.

Principal component analysis of minimal excitatory postsynaptic potentials.

'Minimal' excitatory postsynaptic potentials (EPSPs) are often recorded from central neurones, specifically for quantal analysis. However the EPSPs may emerge from activation of several fibres or transmission sites so that formal quantal analysis may give false results. Here we extended application of the principal component analysis (PCA) to minimal EPSPs. We tested a PCA algorithm and a new graphical 'alignment' procedure against both simulated data and hippocampal EPSPs. Minimal EPSPs were recorded before and up to 3.5 h following induction of long-term potentiation (LTP) in CA1 neurones. In 29 out of 45 EPSPs, two (N=22) or three (N=7) components were detected which differed in latencies, rise time (Trise) or both. The detected differences ranged from 0.6 to 7.8 ms for the latency and from 1.6-9 ms for Trise. Different components behaved differently following LTP induction. Cases were found when one component was potentiated immediately after tetanus whereas the other with a delay of 15-60 min. The immediately potentiated component could decline in 1-2 h so that the two components contributed differently into early (< 1 h) LTP1 and later (1-4 h) LTP2 phases. The noise deconvolution techniques was applied to both conventional EPSP amplitudes and scores of separate components. Cases are illustrated when quantal size (upsilon) estimated from the EPSP amplitudes increased whereas upsilon estimated from the component scores was stable during LTP1. Analysis of component scores could show apparent double-fold increases in upsilon which are interpreted as reflections of synchronized quantal releases. In general, the results demonstrate PCA applicability to separate EPSPs into different components and its usefulness for precise analysis of synaptic transmission.

[Mechanism of hemodynamic effects of right cardiac shunting]. / K mekhanizmu gemodinamicheskikh éffektov pravostoronnego shuntirovaniia serdtsa.

The impact of right cardiac shunting on the hemodynamic changes of the cardiovascular system was examined in acute experiments on intact dogs. The ventricular pacemaker (VP) which is a "Modul" Russian model was connected by the scheme: right ventricle-pulmonary artery. The first 20 minutes of VP operation in the complete right ventricular unloading mode resulted in increased left cardiac contraction force and rate and in elevated vascular pressure. The subsequent functioning of VP was followed by a reduction in positive inotropic effects. The changes occurring in left ventricular function and vascular tone were associated with the inclusion of mechanisms responsible for intracardiac nervous regulation to blood filling changes of the right heart due to their shunting. A fall in right ventricular pressure was perceived by myocardial mechanic receptors, by stimulating the adrenergic neurons of the intracardiac nervous system whose action is short and effected from one state to another in the transitional period.

Atropine inhibits associative potentiation in the hippocampus.

The effects of the muscarinic receptor blocker atropine on associative long-term potentiation were studied in the CA1 region of the rat hippocampus. Focal excitatory post-synaptic potentials (EPSP) were recorded in living slices. Local application of atropine (0.1 mM) significantly inhibited associative long-term potentiation (40-60 min after tetanization) in the "weak" input (str. radiatum 128 +/- 10% compared with 168 +/- 9% in controls). Long-term potentiation at the "strong" input (str. oriens) was increased relative to the control (from 137 +/- 13% to 158 +/- 4%). These results indicate that endogenous acetylcholine aids the development of long-term potentiation in synapses with low levels of activation.

Noise deconvolution based on the L1-metric and decomposition of discrete distributions of postsynaptic responses.

A statistical approach to analysis of amplitude fluctuations of postsynaptic responses is described. This includes (1) using a L1-metric in the space of distribution functions for minimisation with application of linear programming methods to decompose amplitude distributions into a convolution of Gaussian and discrete distributions; (2) deconvolution of the resulting discrete distribution with determination of the release probabilities and the quantal amplitude for cases with a small number (< 5) of discrete components. The methods were tested against simulated data over a range of sample sizes and signal-to-noise ratios which mimicked those observed in physiological experiments. In computer simulation experiments, comparisons were made with other methods of 'unconstrained' (generalized) and constrained reconstruction of discrete components from convolutions. The simulation results provided additional criteria for improving the solutions to overcome 'over-fitting phenomena' and to constrain the number of components with small probabilities. Application of the programme to recordings from hippocampal neurones demonstrated its usefulness for the analysis of amplitude distributions of postsynaptic responses.

Changes in paired-pulse facilitation correlate with induction of long-term potentiation in area CA1 of rat hippocampal slices.

The phenomenon of long-term potentiation is widely used as an experimental model of memory. An approach that has been used to study its underlying mechanisms is to analyse its interaction with presynaptic paired-pulse facilitation. Several studies found no evidence for an interaction in the CA1 hippocampal area, whereas other data, for example from quantal analysis, suggested that presynaptic mechanisms contribute to the maintenance of long-term potentiation. In the present study, initial slopes of field potentials in area CA1 were measured in rat hippocampal slices. "Conventional" long-term potentiation was induced by high-frequency (100 Hz) afferent tetanization of the testing input. "Associative" long-term potentiation was induced by combining lower frequency (40 Hz) tetanization of a testing input with high-frequency tetanization of a second input. The paired-pulse facilitation ratio decreased in the majority of experiments in which long-term potentiation was induced conventionally, but it decreased, increased or did not change after inducing associative potentiation. Decreases in the paired-pulse facilitation correlated inversely with the initial (pre-tetanic) facilitation ratio. A more detailed regression analysis suggests that this correlation results from two other correlations: (i) that between changes in paired-pulse facilitation and the magnitude of long-term potentiation, and (ii) that between initial paired-pulse facilitation and the magnitude of long-term potentiation. The first correlation prevailed during the initial 10 min following tetanization, while the second prevailed 40-60 min later. A post-tetanic decrease in paired-pulse facilitation is evidence for an involvement of presynaptic mechanisms in the maintenance of long-term potentiation. The lack of significant changes in some studies could be due to the inclusion in the analyses of experiments with long-term potentiation of small magnitude, in which changes in paired-pulse facilitation ratios would have been inconsistent. The present study suggests that the early (10-20 min) and late (40-50 min) phases of long-term potentiation were mediated by different mechanisms, with a mixture of these mechanisms during the intermediate period. On the basis of the present and previous studies, the following scheme of involvement of several mechanisms in long-term potentiation maintenance is proposed. The early phase includes two major mechanisms: an increase in the probability of transmitter release, leading to an apparent increase in the number of effective release sites, and an increase in efficacy of one transmitter quantum, probably due to an increased number of postsynaptic receptors. The later phase of long-term potentiation is attributed to an increase in the number of transmitter zones, presumably due to structural modifications.

[Atropine suppresses associative potentiation in the hippocampus]. / Atropin podavliaet assotsiativnuiu potentsiatsiiu v gippokampe.

The effect of the muscarinic antagonist atropine on associative long-term potentiation of the CA1 population EPSPs was studied in rat hippocampal slices. Local application of atropine (10(-4) M) significantly suppressed associative long-term potentiation (40-60 min after the tetanization) in a <> input (str. Radiatum 128 +/- 10% vs 168 +/- 9% in control, p < 0.03). Long term potentiation in the <> input itself was not suppressed and even tended to be enhanced by atropine (str. Oriens 158 +/- 4% vs 137 +/- 13 during control). The results suggest that synaptically released endogenous acethylcholine supports induction of long-term potentiation in the synapses with a low level of activation.

Atropine suppresses associative LTP in the CA1 region of rat hippocampal slices.

The effect of the muscarinic antagonist atropine on associative long-term potentiation (aLTP) of the CA1 population EPSPs was studied in rat hippocampal slices. Local application of atropine (10(-4) M) significantly suppressed aLTP in a 'weak' radiatum input, measured 40-60 min after the tetanization (128 +/- 10% vs. 168 +/- 9% during control, P < 0.03) provided that a 'strong' supporting tetanization was applied to the stratum oriens. LTP in the 'strong' stratum oriens input itself was not suppressed and even tended to be enhanced by atropine (158 +/- 4% vs. 137 +/- 13 during control). The results suggest that synaptically released endogenous acetylcholine supports induction of aLTP in the 'weak' input and does not support induction of homosynaptic LTP in the 'strong' input. A possible physiological role of the cholinergic neuromodulatory system in the hippocampal CA1 region hence could consist in 'equalising' LTP-like changes of synaptic conductivity in pathways with high and low level of activity.

Adaptation of Bacillus FTU and Escherichia coli to alkaline conditions: the Na(+)-motive respiration.

Mechanisms of Na+ transport into the inside-out subcellular vesicles of alkalo- and halotolerant Bacillus FTU and of Escherichia coli grown at different pH have been studied. Both microorganisms growing at pH 7.5 are shown to possess a system of the respiration-dependent Na+ transport which (i) is inhibited by protonophorous uncoupler, by delta pH-discharging agent diethylammonium (DEA) acetate, by micromolar cyanide arresting the H(+)-motive respiratory chain, and by amiloride, and (ii) is resistant to the Na+/H+ antiporter monensin and to Ag+, inhibitor of the Na(+)-motive respiratory chain. Growth at pH 8.6 strongly changes the activator and inhibitor pattern. Now (1) protonophore stimulates the Na+ transport, (2) DEA acetate is without effect in the absence of protonophore and is stimulating in its presence, (3) amiloride and low cyanide are ineffective, (4) monensin and Ag+ completely arrest the Na+ accumulation in the vesicles. Independent of pH of the growth medium, (a) valinomycin is stimulatory for the Na+ transport, (b) Na+ ionophore ETH 157 is inhibitory and, (c) Na+ transport can be supported by NADH----fumarate as well as by ascorbate (TMPD)----O2 electron transfers. Growth at alkaline pH results in the appearance of ascorbate (TMPD) oxidation resistant to low and sensitive to high cyanide concentrations. These relationships are in agreement with the concept (Skulachev, V.P. (1984) Trends Biochem. Sci. 9, 483-485) that adaptation to alkaline conditions in bacteria growing in the high [Na+] media causes substitution of Na+ for H+ as a coupling ion. The obtained data indicate that under alkaline conditions, Na+ can be pumped from the cell by the Na(+)-motive respiratory chain with neither H(+)-motive respiration nor the Na+/H+ antiporter involved. In the Na(+)-motive respiratory chain of Bac. FTU or E. coli, two Na+ pumps are localized, one in its initial and the other in its terminal spans.

[Assessment of the functional status of the left and right ventricles in the intact and damaged heart during volume overload]. / Otsenka funktsional'nogo sostoianiia levogo i pravogo zheludochkov intaktnogo i povrezhdennogo serdtsa v usloviiakh ob"emnoi peregruzki.

Changes in hemodynamics, reserve capacities of intact and damaged myocardium have been studied, with the left and right ventricular functions during mounting volume load assessed separately. It has been shown that exclusion of the part of left ventricular myocardium from contraction deteriorated considerably the pump and contractile function not only of the left but also of the right intact heart ventricle. Heart failure in volume overload manifests itself primarily in the right ventricle and depends on the functional heart reverse on the whole: in animals with partial injury of left ventricular myocardium heart failure developed in much lower volume loads than in intact animals. In circulation volume overload, left or right ventricular stroke work index to preload or filling pressure in the corresponding heart chamber ratio was most adequate and prognostically valuable for the detection of latent heart failure.

[Appraisal of the status of metabolism in myocardial layers in acute left-ventricular insufficiency using data from radionuclide examination methods]. / Otsenka sostoianiia metabolizma v sloiakh miokarda pri ostroi levozheludochkovoi nedostatochnosti po dannym radionuklidnykh metodov issledovaniia.

Experiments were conducted on 35 mongrel dogs. It was demonstrated in series I experiments that exclusion of a 14.5% myocardial area from the heart mass induces acute left-ventricular insufficiency manifested by authentic decrease of the cardiac ejection and left-ventricular output index and a 22% increase of pressure in the left atrium and pulmonary artery. The left-ventricular coronary blood flow and the blood flow in the myocardial layers of the right and left ventricles and in the interventricular septum increased significantly as compared to the control values. Distal to the area of the necrosis, however, the layer-by-layer flow of the blood in the left ventricle had a marked tendency to increase. In series II experiments, in myocardial necrosis the volume load, which was equal to one half and one initial circulation volume, mobilized the heart contractile activity, which led to increase of the coronary blood flow which was less than the initial level in series I experiments. Analysis of layer-by-layer blood flow after a volume load equal to one initial circulation volume showed it to be markedly increased in all layers of the left-ventricular anterior wall above and below the necrotic area, in the left-ventricular posterior wall, in the interventricular septum, and in the right-ventricular anterior wall. The increase of 201Tl-chloride accumulation in the myocardial layers under the studied conditions is evidently linked with activation of metabolic processes responsible for increase of heart contractile activity and, consequently, for increased uptake of 201Tl from circulation by the intact cardiomyocytes rather than with change in redistribution of the blood flow and its increase.