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PubMed2001

Spatial learning deficits without hippocampal neuronal loss in a model of early-onset epilepsy.

Studies were undertaken to examine the effects recurrent early-life seizures have on the ability of rats to acquire spatial memories in adulthood. A minute quantity of tetanus toxin was injected unilaterally into the hippocampus on postnatal day 10. Within 48 h, rats developed recurrent seizures that persisted for 1 week. Between postnatal days 57 and 61, rats were trained in a Morris water maze. Toxin-injected rats were markedly deficient in learning this task. While these rats showed gradual improvement in escape latencies over 20 trials, their performance always lagged behind that of controls. Poor performance could not be explained by motor impairments or motivational difficulties since swimming speed was similar for the groups. Only eight of 16 toxin-injected animals showed focal interictal spikes in the hippocampus during electroencephalographic recordings. This suggests that learning deficiencies and chronic epilepsy may be independent products of recurrent early-life seizures. A quantitative analysis of hippocampus revealed a significant decrease in neuronal density in stratum pyramidale of experimental rats. However, the differences were largely explained by a concomitant increase in the area of stratum pyramidale. Studies of glial fibrillary acidic protein expression and spread of horseradish peroxidase-conjugated tetanus toxin in the hippocampus suggest that the dispersion of cell bodies in stratum pyramidale can neither be explained by a reactive gliosis nor the direct action of the toxin itself. Taken together, we suggest that recurrent seizures beginning in early life can lead to a significant deficiency in spatial learning without ongoing hippocampal synchronized network discharging or a substantial loss of hippocampal pyramidal cells.

Action Potentials↗
PubMed1983

[Properties of spreading depression during different phases of cyclic excitation of the cerebral cortex in the rat].

Properties of cortical spreading depression (SD) were studied at different phases of cyclic excitation occurring in the rat neocortex under low-frequency electrostimulation. SD waves appeared in the cortex spontaneously or were elicited by KCL microinjection. It is shown that the state preventing penetration of SD waves into the stimulated area (blockade of SD) developed during each cycle of excitation. The spatial extent of the SD blockade depended on the degree of excitation generalization over cerebral cortex. After the end of excitation the ability of the stimulated cortical area to conduct SD waves restored. In the intervals between the excitation cycles the SD waves duration was on the average half as long as before stimulation. The development of SD blockade during active phase and restoration of SD waves in the intervals confirms a hypothesis that K+-Na+ pump activation prevents the propagation of SD.

Animals↗
PubMed2004

Visual word recognition: the first half second.

We used magnetoencephalography (MEG) to map the spatiotemporal evolution of cortical activity for visual word recognition. We show that for five-letter words, activity in the left hemisphere (LH) fusiform gyrus expands systematically in both the posterior-anterior and medial-lateral directions over the course of the first 500 ms after stimulus presentation. Contrary to what would be expected from cognitive models and hemodynamic studies, the component of this activity that spatially coincides with the visual word form area (VWFA) is not active until around 200 ms post-stimulus, and critically, this activity is preceded by and co-active with activity in parts of the inferior frontal gyrus (IFG, BA44/6). The spread of activity in the VWFA for words does not appear in isolation but is co-active in parallel with spread of activity in anterior middle temporal gyrus (aMTG, BA 21 and 38), posterior middle temporal gyrus (pMTG, BA37/39), and IFG.

Adult↗
PubMed1999

Cell membrane orientation visualized by polarized total internal reflection fluorescence.

In living cells, variations in membrane orientation occur both in easily imaged large-scale morphological features, and also in less visualizable submicroscopic regions of activity such as endocytosis, exocytosis, and cell surface ruffling. A fluorescence microscopic method is introduced here to visualize such regions. The method is based on fluorescence of an oriented membrane probe excited by a polarized evanescent field created by total internal reflection (TIR) illumination. The fluorescent carbocyanine dye diI-C(18)-(3) (diI) has previously been shown to embed in the lipid bilayer of cell membranes with its transition dipoles oriented nearly in the plane of the membrane. The membrane-embedded diI near the cell-substrate interface can be fluorescently excited by evanescent field light polarized either perpendicular or parallel to the plane of the substrate coverslip. The excitation efficiency from each polarization depends on the membrane orientation, and thus the ratio of the observed fluorescence excited by these two polarizations vividly shows regions of microscopic and submicroscopic curvature of the membrane, and also gives information regarding the fraction of unoriented diI in the membrane. Both a theoretical background and experimental verification of the technique is presented for samples of 1) oriented diI in model lipid bilayer membranes, erythrocytes, and macrophages; and 2) randomly oriented fluorophores in rhodamine-labeled serum albumin adsorbed to glass, in rhodamine dextran solution, and in rhodamine dextran-loaded macrophages. Sequential digital images of the polarized TIR fluorescence ratios show spatially-resolved time-course maps of membrane orientations on diI-labeled macrophages from which low visibility membrane structures can be identified and quantified. To sharpen and contrast-enhance the TIR images, we deconvoluted them with an experimentally measured point spread function. Image deconvolution is especially effective and fast in our application because fluorescence in TIR emanates from a single focal plane.

Adsorption↗
PubMed2003

Differential spatial and temporal phosphorylation of the visual receptor, rhodopsin, at two primary phosphorylation sites in mice exposed to light.

Phosphorylation of rhodopsin critically controls the visual transduction cascade by uncoupling it from the G-protein transducin. The kinase primarily responsible for this phosphorylation is rhodopsin kinase, a substrate-regulated kinase that phosphorylates light-activated rhodopsin. Protein kinase C has been implicated in controlling the phosphorylation of both light-activated and dark-adapted rhodopsin. Two of the major rhodopsin phosphorylation sites in vivo, Ser(334) and Ser(338), are effective protein kinase C phosphorylation sites in vitro, while the latter is preferentially phosphorylated by rhodopsin kinase in vitro. Using phosphospecific antibodies against each of these two sites, we show that both sites are under differential spatial and temporal regulation. Exposure of mice to light results in rapid phosphorylation of Ser(338) that is evenly distributed along the rod outer segment. Phosphorylation of Ser(334) is considerably slower, begins at the base of the rod outer segment, and spreads to the top of the photoreceptor over time. In addition, we show that phosphorylation of both sites is abolished in rhodopsin kinase(-/-) mice, revealing an absolute requirement for rhodopsin kinase to phosphorylate rhodopsin. This requirement may reflect the need for priming phosphorylations at rhodopsin kinase sites allowing for subsequent phosphorylation by protein kinase C at Ser(334). In this regard, treatment of mouse retinas with phorbol esters results in a 4-fold increase in phosphorylation on Ser(334), with no significant effect on the phosphorylation of Ser(338). Our results are consistent with light triggering rapid priming phosphorylations of rhodopsin by rhodopsin kinase, followed by a slower phosphorylation on Ser(334), which is regulated by protein kinase C.

Amino Acid Sequence↗
PubMed1999

Radial localization of inositol 1,4,5-trisphosphate-sensitive Ca2+ release sites in Xenopus oocytes resolved by axial confocal linescan imaging.

The radial localization and properties of elementary calcium release events ("puffs") were studied in Xenopus oocytes using a confocal microscope equipped with a piezoelectric focussing unit to allow rapid (>100 Hz) imaging of calcium signals along a radial line into the cell with a spatial resolution of <0.7 micrometer. Weak photorelease of caged inositol 1,4,5-trisphosphate (InsP3) evoked puffs arising predominantly within a 6-micrometer thick band located within a few micrometers of the cell surface. Approximately 25% of puffs had a restricted radial spread, consistent with calcium release from a single site. Most puffs, however, exhibited a greater radial spread (3.25 micrometer), likely involving recruitment of radially neighboring release sites. Calcium waves evoked by just suprathreshold stimuli exhibited radial calcium distributions consistent with inward diffusion of calcium liberated at puff sites, whereas stronger flashes evoked strong, short-latency signals at depths inward from puff sites, indicating deep InsP3-sensitive stores activated at higher concentrations of InsP3. Immunolocalization of InsP3 receptors showed punctate staining throughout a region corresponding to the localization of puffs and subplasmalemmal endoplasmic reticulum. The radial organization of puff sites a few micrometers inward from the plasma membrane may have important consequences for activation of calcium-dependent ion channels and "capacitative" calcium influx. However, on the macroscopic (hundreds of micrometers) scale of global calcium waves, release can be considered to occur primarily within a thin, essentially two-dimensional subplasmalemmal shell.

Animals↗
PubMed1996

Utilizing latency for object recognition in real and artificial neural networks.

A consistent analysis of a visual scene requires the recognition of different objects. In vertebrate brains this could be achieved by synchronization of the activity of disjunct nerve cell assemblies. During such a process cross-talk between spatially adjacent image parts occurs, preventing efficient synchronization. Temporal differences, naturally introduced by stimulus latencies in every sensory system, were utilized in this study to counteract this effect and strongly improve network performance. To this end in our model the image is 'spread out' in time as a function of contrast-dependent visual latencies, and synchronization of cell assemblies occurs without mutual disturbance. The network model requires a direct link between visual latencies and the onset of synchronous oscillations in cortical cells. This was confirmed experimentally.

Cognition↗
PubMed1996

Interactions between electrical activity and cortical microcirculation revealed by imaging spectroscopy: implications for functional brain mapping.

Modern neuroimaging techniques use signals originating from microcirculation to map brain function. In this study, activity-dependent changes in oxyhemoglobin, deoxyhemoglobin, and light scattering were characterized by an imaging spectroscopy approach that offers high spatial, temporal, and spectral resolution. Sensory stimulation of cortical columns initiates tissue hypoxia and vascular responses that occur within the first 3 seconds and are highly localized to individual cortical columns. However, the later phase of the vascular response is less localized, spreading over distances of 3 to 5 millimeters.

Animals↗
PubMed2001

Structure and substitutions in fluorapatite.

Fluorapatite, Ca10(PO4)6F2. is a widely spread form of calcium phosphate present particularly in biological material. Human hard tissues contain crystals structurally related to apatite. Fluoride can be found in various natural sources and is also used for its beneficial action in caries prevention. Fluorapatite belongs to the spatial group P6(3/m) (C(6h)2) and consists of 3 ions: F-, Ca2+, PO4(3-). In the present paper, we have carried out a crystallographic study of the fluorapatite structure and of the changes induced by the substitutions. The fluorapatite structure and the presence of a large number of ionic bonds make fluorapatite a very suitable host for many substituents, some of them harmless for the human organism, some not. According to the substitution site, we can describe four types of substitution. The F- substitution, also called Type A substitution, is the main one, and the best known. Only the Ca2+ substitution implies changes in the crystal structure. However, some questions remain, in particular for the PO4(3-) substitution, which is the main substitution present in the biological calcium phosphates.

Journal Article↗
PubMed1995

Cardiac muscle cell interaction: from microanatomy to the molecular make-up of the gap junction.

Electrical coupling between cardiac muscle cells is mediated by specialised sites of plasma membrane interaction termed gap junctions, one of three types of intercellular junction of the cardiac intercalated disk. Gap junctions consist of clusters of plasma membrane channels directly linking the cytoplasmic compartments of neighbouring cells. Gap-junctional channels are constructed from connexins, a multigene family of conserved proteins. The principal connexin isoform of the mammalian heart is connexin43; other connexins, notably connexin40, connexin45 and connexin37, are also expressed but in smaller quantities. Antibodies directed against unique sequences of these molecules allow investigation of the role of gap junctions and their component connexins in relation to the electrophysiological properties of the healthy and diseased heart. Confocal laser scanning microscopy of working ventricular myocytes immunolabelled with anticonnexin43 antibodies permits highly sensitive detection of gap junctions, allowing detailed analysis of the spatial distribution of the conduction pathways from the level of the cell to that of the tissue as a whole. Gap junction distribution, number and regional variations in the type of connexin expressed all contribute to the uniform anisotropic pattern of impulse spread characteristic of normal myocardium and the orderly, sequential contraction of the cardiac chambers. Connexin40 is preferentially expressed by myocytes of the atrioventricular conduction system and represents a specialisation facilitating fast conduction, allowing rapid distribution of the impulse throughout the working ventricle. Two major abnormalities in connexin43 gap junctions are detected in human ischaemic heart disease. First, at border zones adjacent to infarct scars, zones which are particularly prone to re-entry arrhythmia, there is marked disruption of the usual ordered distribution pattern of gap junctions. Second, a widespread downregulation of connexin43 gap junctions occurs in myocardium distant from the infarct, a change that is also found in the hypertrophic (non-ischaemic) heart. Consequent localised heterogeneous conduction and reduced conduction velocity provide an explanation for the genesis of re-entry arrhythmias. A current working hypothesis is that reduction in connexin43 gap junctions is a general pathogenetic feature of cardiac disease which predisposes the heart to arrhythmia, and that this reduction may form part of a wider pattern of alteration in the levels of other connexin isoforms.

Animals↗
PubMed1994

Evidence that cell surface beta 1,4-galactosyltransferase spontaneously galactosylates an underlying laminin substrate during fibroblast migration.

beta 1,4-Galactosyltransferase is unusual among the glycosyltransferases in that a subpopulation exists on the cell surface in addition to its traditional biosynthetic location within the Golgi complex. On the cell surface, galactosyltransferase is expressed in spatially restricted, cell type-specific domains, where it functions as a receptor for extracellular oligosaccharide ligands during selected cellular interactions. For example, galactosyltransferase is found on the leading and trailing edges of migrating cells, where it facilitates lamellipodia formation and cell spreading by binding to specific N-linked oligosaccharides within laminin. Although the ability of galactosyltransferase to serve as a laminin receptor is well documented, it is unclear whether it functions solely in a lectin-like capacity to bind laminin glycoside ligands or uses its intrinsic catalytic activity to release itself from and modify its oligosaccharide substrate. In this study, we determined whether cell surface galactosyltransferase spontaneously galactosylates laminin matrices during cell migration using endogenous galactose donors. Cells were prelabeled with [3H]galactose, washed, and transferred in small clusters onto laminin matrices. The prelabeled cells migrated out from the cell cluster, during which time they deposited covalently bound [3H]galactose residues onto the laminin matrix. The degree of galactosylation was both laminin- and time-dependent and required actively migrating, intact cells. The radioactivity released from the 3H-galactosylated laminin by acid hydrolysis comigrated with authentic galactose standards on paper chromatography. In parallel assays, there was no radioactivity deposited on laminin matrices when cells were prelabeled with [3H]fucose or [3H]leucine. Furthermore, [3H]galactosylation was dependent upon galactosyltransferase-mediated cell migration, since prelabeled cells did not deposit [3H]galactose when migrating on fibronectin, upon which migration is integrin-dependent and galactosyltransferase-independent. These results raise the possibility that galactosyltransferase functions catalytically during cell migration, either to dissociate from its oligosaccharide ligand and/or to modify the extracellular matrix.

3T3 Cells↗
PubMed2003

An improved analytical detector response function model for multilayer small-diameter PET scanners.

The optimization of spatial resolution is a critical consideration in the design of small-diameter positron emission tomography (PET) scanners for animal imaging, and is often addressed with Monte Carlo simulations. As a faster and simpler solution, we have developed a new analytical model of the PET detector response function, and implemented the model for a small single-slice, multilayer PET scanner. The accuracy of the model has been assessed by comparison with both Monte Carlo simulations and experimental measurements published in the literature. Results from the analytical model agreed well with the Monte Carlo method, being noise free and two to three orders of magnitude faster. The only major discrepancy was a slight underestimation of the width of the point spread function by the analytical method as inter-crystal scatter is neglected. We observed good agreement between the predictions of the model and experimental measurements. For two large-diameter scanners additional discrepancies were seen due to photon acollinearity, which is not considered in the model. We have shown that the simple and fast analytical detector response function model can provide accurate estimates of spatial resolution for small-diameter PET scanners, and could be a useful tool for several applications, complementing or cross-validating other simulation methods.

Algorithms↗
PubMed2006

Acute and chronic changes in aquaporin 4 expression after spinal cord injury.

The effect of spinal cord injury (SCI) on the expression levels and distribution of water channel aquaporin 4 (AQP4) has not been studied. We have found AQP4 in gray and white matter astrocytes in both uninjured and injured rat spinal cords. AQP4 was detected in astrocytic processes that were tightly surrounding neurons and blood vessels, but more robustly in glia limitans externa and interna, which were forming an interface between spinal cord parenchyma and cerebrospinal fluid (CSF). Such spatial distribution of AQP4 suggests a critical role that astrocytes expressing AQP4 play in the transport of water from blood/CSF to spinal cord parenchyma and vice versa. SCI induced biphasic changes in astrocytic AQP4 levels, including its early down-regulation and subsequent persistent up-regulation. However, changes in AQP4 expression did not correlate well with the onset and magnitude of astrocytic activation, when measured as changes in GFAP expression levels. It appears that reactive astrocytes began expressing increased levels of AQP4 after migrating to the wound area (thoracic region) two weeks after SCI, and AQP4 remained significantly elevated for months after SCI. We also showed that increased levels of AQP4 spread away from the lesion site to cervical and lumbar segments, but only in chronically injured spinal cords. Although overall AQP4 expression levels increased in chronically-injured spinal cords, AQP4 immunolabeling in astrocytic processes forming glia limitans externa was decreased, which may indicate impaired water transport through glia limitans externa. Finally, we also showed that SCI-induced changes in AQP4 protein levels correlate, both temporally and spatially, with persistent increases in water content in acutely and chronically injured spinal cords. Although correlative, this finding suggests a possible link between AQP4 and impaired water transport/edema/syringomyelia in contused spinal cords.

Animals↗
PubMed2002

[Proximal interlocking of humeral intramedullary nails and risk of axillary nerve injury].

QUESTION: Possible injuries to the axillary nerve are criticised in antegrade and retrograde endomedullary nailing of the humeral shaft during proximal interlocking. Clinical experience seems not to support the theory of a high risk of nerve injury. The real risk although remains unknown under anatomical-morphological aspects. MATERIAL AND METHODS: Eight complete human cadaveric shoulder-arm regions with original soft-tissue coverage had been used. Four times the unreamed humeral nail (UHN) had been inserted retrograde, and four times antegrade, distributed in each pair. Antegrade interlocking was performed after skin incision and spreading of soft tissues through the aiming devices. This involved one oblique bolt from lateral to medial. Retrograde proximal interlocking was performed under image intensifier. This involved triple interlocking, once dorso-ventrally and twice latero-medially. The incision paths have been marked. After preparation of the nerve stem in the lateral axillary portal the different branches have been searched and exposed. The spatial relations of bolts and nerve branches have been measured and the insertion path of the bolts has been revised, finally lesions of nerve structures have been documented. RESULTS: We found the latero-medially inserted bolt heads of the retrograde approach and the oblique bolt head of the antegrade approach being placed in a safe distance from the medio-dorsally positioned stem of the axillary nerve. On the other hand the dorsoventrally inserted bolt head (retrograde approach) showed in most cases a very tight relation to the nerve stem. Exploring the axillary nerve and its branches showed in no case a direct nerve lesion. CONCLUSIONS: We suggest to perform only a sharp cutaneous incision and then to prepare the muscle only by careful spreading until touching bone, with tissue retraction during drilling. In retrograde nailing the dorso-ventral bolt should only be used in extreme proximal fractures.

Axilla↗
PubMed2004

Mathematical modelling of an ischemic stroke: an integrative approach.

Understanding the mechanisms and the time and spatial evolution of penumbra following an ischemic stroke is crucially important for developing therapeutics aimed at preventing this area from evolving towards infarction. To help in integrating the available data, we decided to build a formal model. We first collected and categorised the major available evidence from animal models and human observations and summarized this knowledge in a flow-chart with the potential key components of an evolving stroke. Components were grouped in ten sub-models that could be modelled and tested independently: the sub-models of tissue reactions, ionic movements, oedema development, glutamate excitotoxicity, spreading depression, NO synthesis, inflammation, necrosis, apoptosis, and reperfusion. Then, we figured out markers, identified mediators and chose the level of complexity to model these sub-models. We first applied this integrative approach to build a model based on cytotoxic oedema development following a stroke. Although this model includes only three sub-models and would need to integrate more mechanisms in each of these sub-models, the characteristics and the time and spatial evolution of penumbra obtained by simulation are qualitatively and, to some extent, quantitatively consistent with those observed using medical imaging after a permanent occlusion or after an occlusion followed by a reperfusion.

Animals↗
PubMed1999

Spatial regulation and surface chemistry control of monocyte/macrophage adhesion and foreign body giant cell formation by photochemically micropatterned surfaces.

A long-standing goal of biomedical device development has been the generation of specific, desired host blood and tissue responses. An approach to meeting this design criteria is precise surface modification that creates micropatterns of distinct physicochemical character to direct cell adhesion and behavior. For this study, poly(ethylene terephthalate) films were coated with poly(benzyl N, N-diethyldithiocarbamate-co-styrene) and sequentially exposed to monomer solutions for photoirradiation. A photomask was placed over different regions to generate micropatterned surfaces with graft polymer stripes of three distinct ionic characters. Human monocytes were cultured on these surfaces to ascertain whether adhesion and fusion of monocytes/macrophages could be controlled. Nonionic polyacrylamide greatly inhibited adhesion and induced clumping of the few monocytes that did adhere. Macrophage adhesion and spreading led to high degrees of interleukin-13 induced foreign body giant cell formation on both the anionic poly(acrylic acid), sodium salt, and benzyl N,N-diethyldithiocarbamate portions of the culture surface. In spite of the highest observed levels of monocyte/macrophage adhesion on cationic poly(dimethylaminopropylacrylamide), methiodide, the adherent cells were not competent to undergo fusion to form foreign body giant cells. These results suggest that inflammatory cell responses may be spatially controlled in a manner that may be ultimately exploited to improve the biocompatibility of medical devices.

Acrylic Resins↗
PubMed2005

Mechanisms of Hedgehog gradient formation and interpretation.

Morphogens are molecules that spread from localized sites of production, specifying distinct cell outcomes at different concentrations. Members of the Hedgehog (Hh) family of signaling molecules act as morphogens in different developmental systems. If we are to understand how Hh elicits multiple responses in a temporally and spatially specific manner, the molecular mechanism of Hh gradient formation needs to be established. Moreover, understanding the mechanisms of Hh signaling is a central issue in biology, not only because of the role of Hh in morphogenesis, but also because of its involvement in a wide range of human diseases. Here, we review the mechanisms affecting the dynamics of Hh gradient formation, mostly in the context of Drosophila wing development, although parallel findings in vertebrate systems are also discussed.

Animals↗
PubMed1987

Simulation of the point spread function for light in tissue by a Monte Carlo method.

We have been able by a Monte Carlo technique to generate the point spread function (PSF) for light in tissue for a generalized range of tissue characteristics. We have demonstrated that these can be described by an equation containing a gaussian, diffusion and exponential term. The PSF equation will allow one to estimate the limits of spatial resolution achievable with near infrared (NIR) imaging systems, and may be used in image deconvolution algorithms. Additionally an equation has been derived describing the average photon pathlength through the tissue. Finally, the light transmission and reflection (backscattering) have been illustrated as functions of scattering and absorption coefficients. These results can be used in attempting to quantify data from non-invasive NIR spectroscopy systems.

Brain↗