Transcriptional regulation of caspases in experimental pneumococcal meningitis.

Apoptosis and necrosis in brain account for neurological sequelae in survivors of bacterial meningitis. In meningitis, several mechanisms may trigger death pathways leading to activation of transcription factors regulating caspases mRNA synthesis. Therefore, we used a multiprobe RNA protection assay (RPA) to examine the expression of 9 caspase-mRNA in the course of experimental Streptococcus pneumoniae meningitis in mouse brain. Caspase-6, -7 and -11 mRNA were elevated 6 hours after infection. 12 hours after infection caspases-1, -2, -8 and -12 mRNA rose. Caspase-14 mRNA was elevated 18 h and caspase-3 mRNA 24 h after infection. In situ hybridization detected caspases-3, -8, -11 and -12 mRNA in neurons of the hippocampal formation and neocortex. Development of sepsis was paralleled by increased transcription of caspases mRNA in the spleen. In TNFalpha-deficient mice all caspases examined were less upregulated, in TNF-receptor 1/2 knockout mice caspases-1, -2, -7, -11 and -14 mRNA were increased compared to infected control animals. In caspase-1 deficient mice, caspases-11, and -12 mRNA levels did not rise in meningitis indicating the necessity of caspase-1 activating these caspases. Hippocampal formations of newborn mice incubated with heat-inactivated S. pneumoniae R6 showed upregulation of caspase-1, -3, -11 and -12 mRNA. These observations suggest a tightly regulated caspases network at the transcriptional level in addition to the known cascade at the protein level.

Organotypic hippocampal cultures. A model of brain tissue damage in Streptococcus pneumoniae meningitis.

Hippocampal slices of newborn rats were exposed to either heat-inactivated Streptococcus pneumoniae R6 (hiR6) equivalent to 10(6) and 10(8) CFU/ml, lipoteichoic acid (LTA) (0.3 microg/ml and 30 microg/ml), peptidoglycans (PG) (0.3, 30, 50 and 100 microg/ml), pneumococcal DNA (pDNA) (0.3 and 30 microg/ml) or medium only (control). Cell injury was examined by Nissl staining, Annexin V and NeuN immunohistochemistry, and quantified by propidium iodide (PI) uptake and by determining neuron-specific enolase (NSE) concentration in the culture medium. Necrotic and apoptotic cell damage occurred in all treatment groups. Overall damage (Nissl and PI staining) was most prominent after hiR6 (10(8) CFU/ml), followed by LTA (30 microg/ml), pDNA (30 microg/ml), and not detectable after PG (30 microg/ml) exposure. PG (100 microg/ml) induced severe damage. Apoptotic cells were most frequent after exposure to LTA and hiR6. Damage in the neuronal cell layers (NeuN, NSE) was most severe after treatment with hiR6 (10(8) CFU/ml), followed by PG (100 microg/ml), pDNA (30 microg/ml), and LTA (30 microg/ml).

Interaction between paired-pulse facilitation and long-term potentiation in area CA1 of guinea-pig hippocampal slices: application of quantal analysis.

The aim of the study was to further specify mechanisms of maintenance of hippocampal long-term potentiation. Previous analysis of excitatory postsynaptic potentials showed increases in quantal content (mean number of neurotransmitter quanta released by every testing pulse) with smaller increases in quantal size (effect of one transmitter quantum) following long-term potentiation induction. Here we recorded intracellularly excitatory postsynaptic potentials from CA1 pyramidal neurons of guinea-pig hippocampal slices after minimal paired-pulse stimulation of monosynaptic inputs. Statistical parameters underlying excitatory postsynaptic potential fluctuations were estimated by a deconvolution procedure using a quantal model. The parameters of excitatory postsynaptic potentials following paired-pulse stimulation were studied before and after induction of long-term potentiation. Under both conditions, paired-pulse facilitation was found to be accompanied by increases in quantal content and quantal size. During long-term potentiation, paired-pulse facilitation of amplitude and quantal content was lower. The respective changes in the paired-pulse facilitation ratios correlated with long-term potentiation magnitude. In contrast, the paired-pulse facilitation of quantal size did not change significantly following long-term potentiation induction. The results are compatible with the existence of two separate mechanisms of long-term potentiation maintenance. They support the suggestion that changes in quantal content are mainly due to presynaptic mechanisms which are shared by long-term potentiation and paired-pulse facilitation. The mechanisms underlying changes in quantal size are of a different nature for long-term potentiation and paired-pulse facilitation. For long-term potentiation they might be located postsynaptically.

Intracellular calcium redistribution accompanies changes in total tissue Na+, K+ and water during the first two hours of in vitro incubation of hippocampal slices.

Changes of total tissue water, Ca, Na and K contents were monitored in whole transverse hippocampal slices of the guinea-pig during the first 2 h of in vitro incubation. A brief, 75% increase in tissue Ca was noted during the initial 15 min of maintenance, in contrast to a permanent increase of sodium and water contents, coupled to simultaneous decrease of potassium level. The rate of tissue Na, K and water changes comprised a rapid phase at the first 10-20 min, parallel with the increase of the tissue Ca content, and a slow phase during the rest of the incubation period. Development of specific morphological alterations, representative of ischemic/hypoxic lesions and a translocation of calcium from cytoplasm to mitochondria and endoplasmic reticulum during slice maintenance, was also detected by electron microscopy. A two-step mechanism might explain the development of a new steady-state total calcium content of slices. in which the cellular Ca2+ uptake at the beginning of incubation, likely triggered by hypoxic/ ischemic trauma of slice preparation, is followed by a balanced Ca2+ influx, extrusion and sequestration (predominantly into mitochondria and endoplasmic reticulum) during maintenance.

Involvement of silent synapses in the induction of long-term potentiation and long-term depression in neocortical and hippocampal neurons.

Changes in the latency of small excitatory postsynaptic potentials were observed in association with induction of long-term modifications of synaptic transmission in slices of rat neocortex and guinea-pig hippocampus. After potentiation response latency decreased in 3/10 cases in the neocortex and in 6/24 cases in the hippocampus, and increased after depression in 4/8 cases in the neocortex. These latency changes could not be attributed to changes in presynaptic fibre excitability, monosynaptic inhibition, release kinetics or activation kinetics of postsynaptic ion channels. We conclude therefore that potentiation led to the activation of previously silent synapses of fast-conducting afferents and depression to the inactivation of previously functional synapses. Thus, neocortical and hippocampal synapses can be in a non-functional state, and regimes that induce long-term potentiation and depression not only change the efficacy of synapses but also alter their functional state.

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.

Stimulus-induced patterns of bioelectric activity in human neocortical tissue recorded by a voltage sensitive dye.

Stimulus-induced pattern of bioelectric activity in human neocortical tissue was investigated by use of the voltage sensitive dye RH795 and a fast optical recording system. During control conditions stimulation of layer I evoked activity predominantly in supragranular layers showing a spatial extent of up to 3000 microm along layer III. Stimulation in white matter evoked distinct activity in infragranular layers with a spatial extent of up to 3000 microm measured along layer V. The mean amplitude of optical signals close to the stimulated sites in layer I and white matter determined 25 ms following the stimulus, decreased by 50% at a lateral distance of approximately 900 microm and 1200 microm, respectively. Velocity of spread along the vertical stimulation axis reached 0.24 m/s in the supragranular layers (layers I to III) and then decreased to 0.09 m/s following layer I activation; stimulation of white matter induced a velocity of spread in layer V of 0.38 m/s, which slowed down to 0.12 m/s when passing the lower border of lamina IV. The horizontal velocities of spread determined from the stimulation site to a lateral distance of 500 microm reached 0.26-0.28 m/s and 0.28-0.35 m/s for layer I and white matter stimulation, respectively. At larger distances velocity of spread decreased. Increased excitability (Mg(2+)-free solution) had no significant effect on the spatio-temporal distribution of evoked activity as compared with control conditions. There were also no obvious differences between the results obtained in slices, which generated spontaneously sharp waves and those which were not spontaneously active. About 30% of the slices (n=7) displayed a greatly different response pattern, which seemed not to be related in a simple way to the stimulation as was the case in the majority of the investigated slices. The activity pattern of those slices appeared atypical in regard to their deviations of the vertical and horizontal extent of activity, to their reduced spatial extent of activity during increased excitability, to their layer-related distribution of activity, and to the appearance of afterdischarges.Concluding, in 30% of the human temporal lobe slices atypical activity pattern occurred which obviously reflect intrinsic epileptiform properties of the resected tissue. The majority of slices showed stereotyped activity pattern without evidence for increased excitability.

Presynaptic calcium transients evoked by paired-pulse stimulation in the hippocampal slice.

The fluorescent dye Calcium Green and optical recording techniques were used to record intracellular Ca2+ transients resulting from paired-pulse stimulation in stratum moleculare of area CA1 in guinea-pig hippocampal slices. Presumed presynaptic calcium transients were recorded while glutamatergic synaptic transmission was blocked by kynurenic acid. Peak responses to paired-pulses (10-160 ms interval) were higher than responses to single pulses of same stimulation strength (42-23% increase). The isolated response to the second pulse, however, was of smaller magnitude in comparison to the first one; the difference in magnitude depended on the interstimulus interval. Thus, the residual presynaptic free calcium concentration may be responsible for paired-pulse facilitation of synaptic transmission in hippocampus. At the same time, a use-dependent inactivation of presynaptic calcium channels may occur.

Algorithm for semi-automatic sorting of objects to specified tissue domains. An aid for co-ordinating morphometric data with identified tissue components.

A personal computer-based technique was developed that reduces the extent of human efforts in obtaining data, like numerical density of profiles on area, describing the distribution of specific features of histological sections, e.g., density of synaptic profiles or of histochemical reaction products in specified tissue compartments. The procedure consists of (i) marking the objects to be counted, (ii) recording the borderlines of (reference) tissue domains of interest, and (iii) allocating the marked objects to the corresponding domains automatically. This automatic sorting of objects into defined tissue domains is achieved by an algorithm operating on two sets of coordinates: (i) coordinates of points that constitute the boundary of tissue domains (perimeter points) and (ii) coordinates of points marking the particular objects to be sorted. The principle of the sorting calculation is to construct 'segments' of the loop by lines parallel to the ordinate which pass through neighbouring perimeter points. The 'items' to be sorted are classified by the sign of marker flags allocated to each point depending on which side of the perimeter segment they are located. Segmentation and classification procedures are sequentially repeated along the entire perimeter of each domain specified by the operator which may result during the procedure in multiple changes of the sign of the flags. The internal or external location of each item finally is represented by the last sign of its flag. Objects, allocated to domains can be counted and processed further for numerical density determination.

Semiquantitative evaluation of histochemically detectable calcium binding in mammalian brain slices.

A method is described for the electron histochemical demonstration of Ca in the central nervous system, based on fixation on Ca-containing paraformaldehyde solution and the subsequent complexing of Ca by ammonium oxalate. The method resulted in highly electron-dense deposits, with good ultrastructural preservation. The Ca content of the deposits was proved by physico-chemical analysis. The high electron density permitted the counting of deposits and thereby an estimation of their numerical density, via planimetry of electron micrographs. Since pre- and postsynaptic localizations could be distinguished on the basis of ultrastructure, this procedure is regarded as a unique semiquantitative method for estimation of the tissue Ca binding of mammalian brain slices.

Neurophysiological analysis of long-term potentiation in mammalian brain.

Long-term potentiation (LTP) is a persistent increase in postsynaptic response following a high-frequency presynaptic activation. Characteristic LTP features, including input specificity and associativity, make it a popular model to study memory mechanisms. Mechanisms of LTP induction and maintenance are briefly reviewed. Increased intracellular Ca2+ concentration is shown to be critical for LTP induction. This increase is believed to be based on Ca2+ influx secondary to activation of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. Existence of other sources of Ca2+ increase and other critical factors is now becoming evident. They include voltage-dependent Ca2+ channels, Ca2+ intracellular stores, metabotropic glutamate receptors, 'modulatory' transmitters. An example of an involvement of voltage-dependent Ca2+ channels is potentiation induced by intracellular depolarizing pulses. LTP can be divided into decremental earlier (E-LTP) and non-decremental late (L-LTP) phases which explains some inconsistencies in studies of LTP mechanisms. E-LTP is suggested to be based on a transient increase in presynaptic release probabilities. A hypothesis is considered which explains L-LTP by suggesting that Ca2+ activates structural changes leading to an increase in the synaptic gap resistance. This enhances positive synaptic electrical feedback and augments release probability. The hypothesis predicts specific morphological changes, synchronous transmitter release of two or several quanta in some central synapses and the amplification of such synchronization following LTP induction. Data are discussed which maintain these predictions.

Spread of epileptiform potentials in the neocortical slice: recordings with voltage-sensitive dyes.

The spread of epileptiform potentials in guinea pig neocortical slices was investigated by use of voltage sensitive dyes and a fast optical recording technique. Epileptiform activity was induced in a perfusion medium containing 10-20 microM bicuculline-methiodide and by single pulse stimulation of layer I or the white matter. The location of minimal and maximal amplitudes, the shape of the potentials at specific sites and the velocity of spread were independent from the specific stimulation site. The expression of epileptiform activity appeared to depend on specific, possibly geometrical, properties of the tissue.

Stimulation-dependent calcium binding sites in the guinea pig hippocampal slice: an electrophysiological and electron microscopic study.

Transverse slices of the hippocampus of guinea pigs were prepared in order to investigate Ca2+ binding sites in CA1. Electrical stimulation (Schaffer collaterals and stratum oriens) combined with different aminopyridine compounds (AP) were used for neuronal activation. With histochemical methods Ca2+ binding sites were identified and localized at the electron microscopic level as electron dense deposits of granular or elongated shape. After electrical stimulation, electron dense deposits of 30-50 nm diameter were spread at low density over all layers of CA1. Electrical stimulation combined with application of aminopyridine compounds led to electron dense deposits of 60-400 nm diameter, mainly restricted to the activated input layers. Deposits were predominantly found at presynaptic sides, with few at dendrites and glial cells. Application of aminopyridine alone led to very few deposits, spread over the total CA1 area. The results indicate that aminopyridines, if combined with electrical stimulation, display a strong presynaptic action, which results in a remarkable Ca2+-translocation at the preterminal and terminal level. On the dendritic side aminopyridines in the concentrations used for the study weakly activate Ca2+ movements.

Immunohistochemical detection of raf protein kinase in cerebral cortical areas of adult guinea pigs and rats.

The raf protooncogenes are the cellular counterparts of the v-raf oncogene expressed by a murine sarcoma virus. The raf protooncogenes encode cytoplasmic serine/threonine-specific protein kinases which can be activated from different growth factor receptors by phosphorylation. The mRNAs of raf protooncogenes are found in a large variety of normal adult tissues, including the central nervous system. As concerns the distribution and localization of their protein products (the raf kinases), very few data are to be found in the literature. This is the first detailed description of their light microscopic localization in neocortical and allocortical areas of rodents. Preembedding immunohistochemical studies were performed on vibratome sections from the brains of adult guinea pigs and albino rats. The localizations of two isoenzymes, raf-1 kinase and B-raf kinase, were studied with the help of isoenzyme-specific polyclonal antibodies. Both of the antibodies detected raf protein-like immunoreactivity in many neurons and scattered glial cells of the sensory neocortex, and the cingular, pyriform, perirhinal and entorhinal allocortical areas. Pyramidal and non-pyramidal cells of Ammon's horn, granule cells of the dentate fascia and the large neurons in the hilar region were immunoreactive, too. The findings indicated that B-raf protein kinase and raf-1 kinase are present almost ubiquitously in the neurons of the investigated cortical structures. The intensity of staining obtained with serial dilutions of the antibodies indicated that the cytoplasmic concentration of B-raf kinase is tended to be higher than that of raf-1 kinase. The present findings suggested that the raf kinases are localized in postsynaptic structures, mainly in dendrites and cell bodies. Their cytosolic localization and their ability to undergo intracellular translocation during activation and phosphorylation raise the possibility that they play a pivotal role in the intracellular signaling of neurons.

Visual pattern evoked responses and blink reflexes in assessment of MS diagnosis. A clinical study of 135 multiple sclerosis/pathol.

VEPs were measured after pattern reversal in 135 MS patients and 30 control subjects. Neurological findings were documented in a standard manner. An extensive ophthalmological examination of all subjects was part of the study. The latency of P2 was abnormally delayed in 82% of the "definite", in 60% of the "probable" and in 65% of the "possible" MS groups respectively. The VEP was more often delayed in relation to ophthalmological disturbances. Changes in the MS classification had to be made in more than 10% of the patients, due to delay of VEP latency. These were patients with a "spinal" form of MS, which is known to create diagnostic problems. Optically and electrically evoked blink reflexes were recorded in 107 MS patients. All patients with mesencephalic lesions had delayed responses of the optically evoked reflex. 74% of the patients with caudal brainstem lesions had delayed latencies of the components of the electrically evoked blink reflex. The blink reflex was delayed in 18 additional patients without brainstem signs. The possibility of delineating clinically silent brainstem lesions by investigating blink reflexes is discussed.

Calcium accumulation by dendritic mitochondria declines along the apical dendrites of pyramidal neurons in area CA1 of guinea pig hippocampal slices.

An electron microscopic histochemical study was performed in stratum radiatum of area CA1 of guinea pig hippocampal slices in order to determine the spatial distribution of a dendritic mitochondrial subpopulation which accumulated calcium during in vitro incubation. A distribution gradient was found along the course of apical dendrites exhibiting the highest density values at the base of the dendrites and decaying to baseline values at about 50 microns distal from the cell body layer. The pronounced calcium accumulation by mitochondria in the proximal apical dendrites was markedly but not completely reduced by blocking L-type Ca-channels. These results (i) support the observation of a clustered distribution of L-type Ca-channels at the base of apical dendrites, (ii) designate these voltage dependent Ca2+ channels as one of the possible routes for calcium influx caused by hypoxia/ischemia induced during slice preparation, and (iii) emphasize the role of mitochondrial calcium sequestering under ischemic/hypoxic conditions.

Partial suppression of GABAA-mediated inhibition induces spatially restricted epileptiform activity in guinea pig neocortical slices.

The spatio-temporal distribution of evoked activity in guinea pig neocortical slices was investigated during partial suppression of gamma-aminobutyric acid (GABA)A-mediated synaptic inhibition with different concentrations of bicuculline. Activity patterns were recorded by use of a voltage-sensitive dye and a fast optical recording technique. At non-epileptogenic concentrations of bicuculline (0.6-2.5 microM), evoked potentials were of longer duration and larger amplitude, but the spatial extent of spread in the horizontal direction was unaffected. At threshold epileptogenic concentrations of bicuculline (1.25-5 microM), spatially restricted epileptiform activity developed at a distance from the stimulation site which was clearly separated from potentials with non-epileptic characteristics close to the stimulation site. It is concluded that, under moderate disinhibition, stimulus-evoked activity has a suppressive effect on spread and development of epileptiform activity, probably through synchronous activation of still-functioning inhibitory circuits.

Increased binding of calcium in the hippocampal slice during long-term potentiation.

Long-term potentiation (LTP) of CA1 pyramidal neurons was induced by tetanic stimulation in the stratum radiatum of hippocampal slices from guinea pigs. Unstimulated and stimulated slices were treated using a histochemical procedure enabling the electron microscopic (EM) visualization of Ca binding sites. Electron-dense, Ca-containing deposits were found in low numbers in unstimulated slices on pre- and postsynaptic sites. In the stratum radiatum of tetanized slices the overall number of deposits as well as the number of deposits in dendrites was clearly increased. The results support the hypothesis that Ca-dependent postsynaptic mechanisms are important for the generation of LTP.

Quantal analysis of paired-pulse facilitation in guinea pig hippocampal slices.

Intracellular excitatory postsynaptic potentials (EPSPs) were recorded in area CA1 of hippocampal slices from guinea pigs. With paired-pulse stimulation of stratum radiatum, the second stimulus (interval of 40-50 ms) produced an EPSP with enlarged amplitude. Two methods of quantal analysis showed an increase in quantal content of the facilitated EPSP and a smaller increase in quantal size. The correlation between amplitudes of the first and second EPSP was usually insignificant. The results favour a presynaptic location of the mechanisms of the paired-pulse facilitation and suggest increases in the average of transmitter quanta released by presynaptic volley as well as increases in the amount of transmitter in each quantum.

Elevation of intracellular calcium content in area CA1 of hippocampus is not directly correlated with the development of long-term potentiation.

Electron microscopic localization of calcium-containing mitochondria in stratum radiatum of CAl of hippocampal slices was performed after (1) low-frequency stimulation, (2) high-frequency stimulation, and (3) blocking N-methyl-D-aspartate (NMDA) receptors during high-frequency stimulation. Dendritic mitochondria containing Ca deposits were found in a narrow band of stratum radiatum 280-350 microns distant from stratum pyramidale. Axonal mitochondria containing Ca deposits were evenly distributed in stratum radiatum. The total number of calcium containing-mitochondria was highest in long-term potentiated slices, and less in slices treated with APV; the lowest values were obtained with low-frequency stimulation.