Multisensory anatomical pathways.

In order to interact with the multisensory world that surrounds us, we must integrate various sources of sensory information (vision, hearing, touch...). A fundamental question is thus how the brain integrates the separate elements of an object defined by several sensory components to form a unified percept. The superior colliculus was the main model for studying multisensory integration. At the cortical level, until recently, multisensory integration appeared to be a characteristic attributed to high-level association regions. First, we describe recently observed direct cortico-cortical connections between different sensory cortical areas in the non-human primate and discuss the potential role of these connections. Then, we show that the projections between different sensory and motor cortical areas and the thalamus enabled us to highlight the existence of thalamic nuclei that, by their connections, may represent an alternative pathway for information transfer between different sensory and/or motor cortical areas. The thalamus is in position to allow a faster transfer and even an integration of information across modalities. Finally, we discuss the role of these non-specific connections regarding behavioral evidence in the monkey and recent electrophysiological evidence in the primary cortical sensory areas.

Evidence for a sodium-induced activation of central neurogenic mechanisms in one-kidney, one-clip renal hypertensive rats

The mechanisms sustaining high blood pressure in conscious one-kidney, one-clip Goldblatt rats were evaluated with the use of SK&F 64139, a phenylethanolamine N-methyltransferase inhibitor capable of crossing the blood-brain barrier and of captopril, an angiotensin converting enzyme inhibitor. The rats were studied 3 weeks after left renal artery clipping and contralateral nephrectomy. During the developmental phase of hypertension, two groups of rats were maintained on a regular salt (RNa) intake, whereas two other groups were given a low salt (LNa) diet. On the day of the experiment, the base-line mean blood pressure measured in the LNa rats (177.4 +/- 5.2 mm Hg, mean +/- S.E., n = 15) was similar to that measured in the RNa rats (178.7 +/- 5.4 mm Hg, n = 16). SK&F 64139 (12.5 mg p.o.) induced a significantly more pronounced (P less than .001) blood pressure decrease in the RNa rats (-25.6 +/- 3.6 mm Hg, n = 8) than in the LNa rats (-4.3 +/- 3.3 mm Hg, n = 7) during a 90-min observation period. On the other hand, captopril (10 mg p.o.) normalized blood pressure in LNa rats (n = 8), but produced only a 13.4 mm Hg blood pressure drop in RNa rats (n = 8). RNa rats treated with SK&F 64139 were found to have decreased phenylethanolamine N-methyltransferase activity by an average 80% in selected brain stem nuclei when compared with nontreated rats. No significant difference in plasma catecholamine levels was found between the RNa and LNa rats. These results suggest that, in this experimental model of hypertension, the sodium ion might increase the model of hypertension, the sodium ion might increase the vasoconstrictor contribution of the sympathetic system via a centrally mediated neurogenic mechanism while at the same time it decreases the renin-dependency of the high blood pressure.

Nuclear triiodothyronine receptor expression is regulated by axon-Schwann cell contact.

Thyroid hormones, which play an important role in the development and regeneration of the nervous system, require the presence of specific nuclear T3 receptors (NT3R). In this study we provide evidence that NT3R expression by Schwann cells was up-regulated in response to a loss of axonal contact in vitro and in vivo. In dorsal root ganglia explant cultures, Schwann cells which accompanied axons (nerve fibres) were devoid of NT3R. When Schwann cells were orphaned from axon contact by axon transection, all the nuclei of these cells displayed NT3R immunoreactivity. Similar results were obtained in situ; in adult rat sciatic nerve, Schwann cells which ensheathed healthy axons never expressed NT3R immunoreactivity. After sciatic nerve transection in vivo the nuclei of Schwann cells deprived of axonal contact displayed a clear NT3R immunoreaction.

Effect of manganese chloride on the neurochemical profile of the rat hypothalamus.

Manganese (Mn(2+))-enhanced magnetic resonance imaging studies of the neuronal pathways of the hypothalamus showed that information about the regulation of food intake and energy balance circulate through specific hypothalamic nuclei. The dehydration-induced anorexia (DIA) model demonstrated to be appropriate for studying the hypothalamus with Mn(2+)-enhanced magnetic resonance imaging. Manganese is involved in the normal functioning of a variety of physiological processes and is associated with enzymes contributing to neurotransmitter synthesis and metabolism. It also induces psychiatric and motor disturbances. The molecular mechanisms by which Mn(2+) produces alterations of the hypothalamic physiological processes are not well understood. (1)H-magnetic resonance spectroscopy measurements of the rodent hypothalamus are challenging due to the distant location of the hypothalamus resulting in limited measurement sensitivity. The present study proposed to investigate the effects of Mn(2+) on the neurochemical profile of the hypothalamus in normal, DIA, and overnight fasted female rats at 14.1 T. Results provide evidence that γ-aminobutyric acid has an essential role in the maintenance of energy homeostasis in the hypothalamus but is not condition specific. On the contrary, glutamine, glutamate, and taurine appear to respond more accurately to Mn(2+) exposure. An increase in glutamine levels could also be a characteristic response of the hypothalamus to DIA.

High-level transgene expression by homologous recombination-mediated gene transfer.

Gene transfer and expression in eukaryotes is often limited by a number of stably maintained gene copies and by epigenetic silencing effects. Silencing may be limited by the use of epigenetic regulatory sequences such as matrix attachment regions (MAR). Here, we show that successive transfections of MAR-containing vectors allow a synergistic increase of transgene expression. This finding is partly explained by an increased entry into the cell nuclei and genomic integration of the DNA, an effect that requires both the MAR element and iterative transfections. Fluorescence in situ hybridization analysis often showed single integration events, indicating that DNAs introduced in successive transfections could recombine. High expression was also linked to the cell division cycle, so that nuclear transport of the DNA occurs when homologous recombination is most active. Use of cells deficient in either non-homologous end-joining or homologous recombination suggested that efficient integration and expression may require homologous recombination-based genomic integration of MAR-containing plasmids and the lack of epigenetic silencing events associated with tandem gene copies. We conclude that MAR elements may promote homologous recombination, and that cells and vectors can be engineered to take advantage of this property to mediate highly efficient gene transfer and expression.

TrkB signaling is required for postnatal survival of CNS neurons and protects hippocampal and motor neurons from axotomy-induced cell death

Newborn mice carrying targeted mutations in genes encoding neurotrophins or their signaling Trk receptors display severe neuronal deficits in the peripheral nervous system but not in the CNS. In this study, we show that trkB (¿/¿) mice have a significant increase in apoptotic cell death in different regions of the brain during early postnatal life. The most affected region in the brain is the dentate gyrus of the hippocampus, although elevated levels of pyknotic nuclei were also detected in cortical layers II and III and V and VI, the striatum, and the thalamus. Furthermore, axotomized hippocampal and motor neurons of trkB (¿/¿) mice have significantly lower survival rates than those of wild-type littermates. These results suggest that neurotrophin signaling through TrkB receptors plays a role in the survival of CNS neurons during postnatal development. Moreover, they indicate that TrkB receptor signaling protects subpopulations of CNS neurons from injury- and axotomy-induced cell death.

Regional and cellular patterns of reelin mRNA expression in the forebrain of the developing and adult mouse

The reelin gene encodes an extracellular protein that is crucial for neuronal migration in laminated brain regions. To gain insights into the functions of Reelin, we performed high-resolution in situ hybridization analyses to determine the pattern of reelin expression in the developing forebrain of the mouse. We also performed double-labeling studies with several markers, including calcium-binding proteins, GAD65/67, and neuropeptides, to characterize the neuronal subsets that express reelin transcripts. reelinexpression was detected at embryonic day 10 and later in the forebrain, with a distribution that is consistent with the prosomeric model of forebrain regionalization. In the diencephalon, expression was restricted to transverse and longitudinal domains that delineated boundaries between neuromeres. During embryogenesis,reelin was detected in the cerebral cortex in Cajal-Retzius cells but not in the GABAergic neurons of layer I. At prenatal stages, reelin was also expressed in the olfactory bulb, and striatum and in restricted nuclei in the ventral telencephalon, hypothalamus, thalamus, and pretectum. At postnatal stages, reelin transcripts gradually disappeared from Cajal-Retzius cells, at the same time as they appeared in subsets of GABAergic neurons distributed throughout neocortical and hippocampal layers. In other telencephalic and diencephalic regions,reelin expression decreased steadily during the postnatal period. In the adult, there was prominent expression in the olfactory bulb and cerebral cortex, where it was restricted to subsets of GABAergic interneurons that co-expressed calbindin, calretinin, neuropeptide Y, and somatostatin. This complex pattern of cellular and regional expression is consistent with Reelin having multiple roles in brain development and adult brain function.

Improved segmentation of deep brain grey matter structures using magnetization transfer (MT) parameter maps.

Basal ganglia and brain stem nuclei are involved in the pathophysiology of various neurological and neuropsychiatric disorders. Currently available structural T1-weighted (T1w) magnetic resonance images do not provide sufficient contrast for reliable automated segmentation of various subcortical grey matter structures. We use a novel, semi-quantitative magnetization transfer (MT) imaging protocol that overcomes limitations in T1w images, which are mainly due to their sensitivity to the high iron content in subcortical grey matter. We demonstrate improved automated segmentation of putamen, pallidum, pulvinar and substantia nigra using MT images. A comparison with segmentation of high-quality T1w images was performed in 49 healthy subjects. Our results show that MT maps are highly suitable for automated segmentation, and so for multi-subject morphometric studies with a focus on subcortical structures.

Cell death in Leishmania induced by stress and differentiation: programmed cell death or necrosis?

Unicellular organisms, such as the protozoan parasite Leishmania, can be stimulated to show some morphological and biochemical features characteristic of mammalian apoptosis. This study demonstrates that under a variety of stress conditions such as serum deprivation, heat shock and nitric oxide, cell death can be induced leading to genomic DNA fragmentation into oligonucleosomes. DNA fragmentation was observed, without induction, in the infectious stages of the parasite, and correlated with the presence of internucleosomal nuclease activity, visualisation of 45 to 59 kDa nucleases and detection of TUNEL-positive nuclei. DNA fragmentation was not dependent on active effector downstream caspases nor on the lysosomal cathepsin L-like enzymes CPA and CPB. These data are consistent with the presence of a caspase-independent cell death mechanism in Leishmania, induced by stress and differentiation that differs significantly from metazoa.

Triiodothyronine has diverse and multiple stimulating effects on expression of the major myelin protein genes.

If the importance of triiodothyronine (T3) on brain development including myelinogenesis has long been recognized, its mechanism of action at the gene level is still not fully elucidated. We studied the effect of T3 on the expression of myelin protein genes in aggregating brain cell cultures. T3 increases the concentrations of mRNA transcribed from the following four myelin protein genes: myelin basic protein (Mbp), myelin-associated glycoprotein (Mag), proteolipid protein (Plp), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (Cnp). T3 is not only a triggering signal for oligodendrocyte differentiation, but it has continuous stimulatory effects on myelin gene expression. Transcription in isolated nuclei experiments shows that T3 increases Mag and Cnp transcription rates. After inhibiting transcription with actinomycin D, we measured the half-lives of specific mRNAs. Our results show that T3 increases the stability of mRNA for myelin basic protein, and probably proteolipid protein. In vitro translation followed by myelin basic protein-specific immunoprecipitation showed a direct stimulatory effect of T3 on myelin basic protein mRNA translation. Moreover, this stimulation was higher when the mRNA was already stabilized in culture, indicating that stabilization is achieved through mRNA structural modifications. These results demonstrate the diverse and multiple mechanisms of T3 stimulation of myelin protein genes.

Upregulation of the GABA transporter GAT-1 in the gracile nucleus in the spared nerve injury model of neuropathic pain.

Neuropathic pain is a major health issue and is frequently accompanied by allodynia (painful sensations in response to normally non-painful stimulations), and unpleasant paresthesia/dysesthesia, pointing to alterations in sensory pathways normally dedicated to the processing of non-nociceptive information. Interestingly, mounting evidence indicate that central glial cells are key players in allodynia, partly due to changes in the astrocytic capacity to scavenge extracellular glutamate and gamma-aminobutyric acid (GABA), through changes in their respective transporters (EAAT and GAT). In the present study, we investigated the glial changes occurring in the dorsal column nuclei, the major target of normally innocuous sensory information, in the rat spared nerve injury (SNI) model of neuropathic pain. We report that together with a robust microglial and astrocytic reaction in the ipsilateral gracile nucleus, the GABA transporter GAT-1 is upregulated with no change in GAT-3 or glutamate transporters. Furthermore, [(3)H] GABA reuptake on crude synaptosome preparation shows that transporter activity is functionally increased ipsilaterally in SNI rats. This GAT-1 upregulation appears evenly distributed in the gracile nucleus and colocalizes with astrocytic activation. Neither glial activation nor GAT-1 modulation was detected in the cuneate nucleus. Together, the present results point to GABA transport in the gracile nucleus as a putative therapeutic target against abnormal sensory perceptions related to neuropathic pain.

The biology of cancer stem cells : part 1: nuclear translocation of CD44 : part 2: IMP2 in glioblastoma cancer stem cells

SummaryCancer stem cells (CSC) are poorly differentiated, slowly proliferating cells, with high tumorigenic potential. Some of these cells, as it has been shown in leukemia, evade chemo- and radiotherapy and recapitulate the tumor composed of CSC and their highly proliferative progeny. Therefore, understanding the molecular biology of those cells is crucial for improvement of currently used anti-cancer therapies.This work is composed of two CSC-related projects. The first deals with CD44, a frequently used marker of CSC; the second involves Imp2 and its role in CSC bioenergetics. PART 1. CD44 is a multifunctional transmembrane protein involved in migration, homing, adhesion, proliferation and survival. It is overexpressed in many cancers and its levels are correlated with poor prognosis. CD44 is also highly expressed by CSC and in many malignancies it is used for CSC isolation.In the present work full-lenght CD44 nuclear localization was studied, including the mechanism of nuclear translocation and its functional role in the nucleus. Full-length CD44 can be found in nuclei of various cell types, regardless of their tumorigenic potential. For nuclear localization, CD44 needs to be first inserted into the cell membrane, from which it is transported via the endocytic pathway. Upon binding to transportinl it is translocated to the nucleus. The nuclear localization signal recognized by transportinl has been determined as the first 20 amino acids of the membrane proximal intracellular domain. Nuclear export of CD44 is facilitated by exportin Crml. Investigation of the function of nuclear CD44 revealed its implication in de novo RNA synthesis.PART 2. Glioblastoma multiforme is the most aggressive and most frequent brain malignancy. It was one of the first solid tumors from which CSC have been isolated. Based on the similarity between GBM CSC and normal stem cells expression of an oncofetal mRNA binding protein Imp2 has been investigated.Imp2 is absent in normal brain as well as in low grade gliomas, but is expressed in over 75% GBM cases and its expression is higher in CSC compared to their more differentiated counterparts. Analysis of mRNA transcripts bound by Imp2 and its protein interactors revealed that in GBM CSC Imp2 may be implicated in mitochondrial metabolism. Indeed, shRNA mediated silencing of protein expression led to decreased mitochondrial activity, decreased oxygen consumption and decreased activity of respiratory chain protein complex I. Moreover, lack of Imp2 severely affected self-renewal and tumorigenicity of GBM CSC. Experimental evidence suggest that GBM CSC depend on mitochondrial oxidative phosphorylation as an energy producing pathway and that Imp2 is a novel regulator of this pathway.RésuméLes cellules cancéreuses souches sont des cellules peu différentiées, à proliferation lente et hautement tumorigénique. Ces cellules sont radio-chimio résistantes et sont capable reformer la tumeur dans sont intégralité, reproduisant l'hétérogénéité cellulaire présent dans la tumeur d'origine. Pour améliorer les therapies antitumorales actuelles il est crucial de comprendre les mécanismes moléculaires qui caractérisent cette sous-population de cellules hautement malignes.Ce travail de thèse se compose de deux projets s'articulant autour du même axe :Le CD44 est une protéine multifonctionnelle et transmembranaire très souvent utilisée comme marqueur de cellules souches tumorales dans différents cancers. Elle est impliquée dans la migration, l'adhésion, la prolifération et la survie des cellules. Lors de ce travail de recherche, nous nous sommes intéressés à la localisation cellulaire du CD44, ainsi qu'aux mécanismes permettant sa translocation nucléaire. En effet, bien que principalement décrit comme un récepteur de surface transmembranaire, le CD44 sous sa forme entière, non clivée en peptides, peut également être observé à l'intérieur du noyau de diverses cellules, quel que soit leur potentiel tumorigénique. Pour passer ainsi d'un compartiment cellulaire à un autre, le CD44 doit d'abord être inséré dans la membrane plasmique, d'où il est transporté par endocytose jusqu'à l'intérieur du cytoplasme. La transportai permet ensuite la translocation nucléaire du CD44 via une « séquence signal » contenue dans les 20 acides aminés du domaine cytoplasmique qui bordent la membrane. A l'inverse, le CD44 est exporté du noyau grâce à l'exportin Crml. En plus des mécanismes décrits ci-dessus, cette étude a également mis en évidence l'implication du CD44 dans la synthèse des ARN, d'où sa présence dans le noyau.Le glioblastome est la plus maligne et la plus fréquente des tumeurs cérébrales. Dans ce second projet de recherche, le rôle de IMP2 dans les cellules souches tumorales de glioblastomes a été étudié. La présence de cette protéine oncofoetale a d'abord été mise en évidence dans 75% des cas les plus agressifs des gliomes (grade IV, appelés glioblastomes), tandis qu'elle n'est pas exprimée dans les grades I à III de ces tumeurs, ni dans le cerveau sain. De plus, IMP2 est apparue comme étant davantage exprimée dans les cellules souches tumorales que dans les cellules déjà différenciées. La baisse de l'expression de IMP2 au moyen de shRNA a résulté en une diminution de l'activité mitochondriale, en une réduction de la consommation d'oxygène ainsi qu'en une baisse de l'activité du complexe respiratoire I.L'inhibition de IMP2 a également affecté la capacité de renouvellement de la population des cellules souches tumorales ainsi que leur aptitude à former des tumeurs.Lors de ce travail de thèse, une nouvelle fonction d'un marqueur de cellules souches tumorales a été mise en évidence, ainsi qu'un lien important entre la bioénergétique de ces cellules et l'expression d'une protéine oncofoetale.

Evidence for segregated and integrative connectivity patterns in the human Basal Ganglia.

Detailed knowledge of the anatomy and connectivity pattern of cortico-basal ganglia circuits is essential to an understanding of abnormal cortical function and pathophysiology associated with a wide range of neurological and neuropsychiatric diseases. We aim to study the spatial extent and topography of human basal ganglia connectivity in vivo. Additionally, we explore at an anatomical level the hypothesis of coexistent segregated and integrative cortico-basal ganglia loops. We use probabilistic tractography on magnetic resonance diffusion weighted imaging data to segment basal ganglia and thalamus in 30 healthy subjects based on their cortical and subcortical projections. We introduce a novel method to define voxel-based connectivity profiles that allow representation of projections from a source to more than one target region. Using this method, we localize specific relay nuclei within predefined functional circuits. We find strong correlation between tractography-based basal ganglia parcellation and anatomical data from previously reported invasive tracing studies in nonhuman primates. Additionally, we show in vivo the anatomical basis of segregated loops and the extent of their overlap in prefrontal, premotor, and motor networks. Our findings in healthy humans support the notion that probabilistic diffusion tractography can be used to parcellate subcortical gray matter structures on the basis of their connectivity patterns. The coexistence of clearly segregated and also overlapping connections from cortical sites to basal ganglia subregions is a neuroanatomical correlate of both parallel and integrative networks within them. We believe that this method can be used to examine pathophysiological concepts in a number of basal ganglia-related disorders.

High-resolution ultrasonography of subretinal structure and assessment of retina degeneration in rat.

The purpose of this work was to evaluate the ability of 80 MHz ultrasonography to differentiate intra-retinal layers and quantitatively assess photoreceptor dystrophy in small animal models. Four groups of 10 RCS rats each (five dystrophic and five controls) were explored at 25, 35, 45 and 55 days post-natal (PN). A series of retina cross-sections were obtained ex vivo from outside intact eyes using an 80 MHz three-dimensional ultrasound backscatter microscope (20-microm-axial resolution). Ultrasound features of normal retina were correlated to those of corresponding histology and thickness measurements of photoreceptor segment and nuclear layers were performed on all groups. To show the ability of 80 MHz ultrasonography to distinguish the retinal degeneration in vivo, one RCS rat was explored at 25 and 55 days post-natal. Ultrasound image of normal retina displayed four distinct layers marked by reflections at neurites/nuclei interfaces and permitted to differentiate the photoreceptor segment and nuclear layers. The backscatter level from the retina was shown to be related to the size, density and organization of the intra-layer structure. Ultrasound thickness measurements highly correlated with histologic measurements. A thinning (p<0.05) of outer nuclear layer (ONL) was detected over time for controls and was thought to be assigned to retina maturation. Retinal degeneration started at PN35 and resulted in a more pronounced ONL thinning (p<0.05) over time. ONL degeneration was accompanied by segment layer thickening (p<0.05) at PN35 and thinning thereafter. These changes may indicate accumulation of outer segment debris at PN35 then progressive destruction. In vivo images of rat intra-retinal structure showed the ability of the method to distinguish the photoreceptor layer changes. Our results indicate that 80 MHz ultrasonography reveals intra-retinal layers and is sensitive to age and degenerative changes of photoreceptors. This technique has great potential to follow-up retinal dystrophy and therapeutic effects in vivo.

New Developments and Applications of the MP2RAGE Sequence - Focusing the Contrast and High Spatial Resolution R1 Mapping.

MR structural T1-weighted imaging using high field systems (>3T) is severely hampered by the existing large transmit field inhomogeneities. New sequences have been developed to better cope with such nuisances. In this work we show the potential of a recently proposed sequence, the MP2RAGE, to obtain improved grey white matter contrast with respect to conventional T1-w protocols, allowing for a better visualization of thalamic nuclei and different white matter bundles in the brain stem. Furthermore, the possibility to obtain high spatial resolution (0.65 mm isotropic) R1 maps fully independent of the transmit field inhomogeneities in clinical acceptable time is demonstrated. In this high resolution R1 maps it was possible to clearly observe varying properties of cortical grey matter throughout the cortex and observe different hippocampus fields with variations of intensity that correlate with known myelin concentration variations.