Efficient gene delivery and selective transduction of astrocytes in the mammalian brain using viral vectors.

Astrocytes are now considered as key players in brain information processing because of their newly discovered roles in synapse formation and plasticity, energy metabolism and blood flow regulation. However, our understanding of astrocyte function is still fragmented compared to other brain cell types. A better appreciation of the biology of astrocytes requires the development of tools to generate animal models in which astrocyte-specific proteins and pathways can be manipulated. In addition, it is becoming increasingly evident that astrocytes are also important players in many neurological disorders. Targeted modulation of protein expression in astrocytes would be critical for the development of new therapeutic strategies. Gene transfer is valuable to target a subpopulation of cells and explore their function in experimental models. In particular, viral-mediated gene transfer provides a rapid, highly flexible and cost-effective, in vivo paradigm to study the impact of genes of interest during central nervous system development or in adult animals. We will review the different strategies that led to the recent development of efficient viral vectors that can be successfully used to selectively transduce astrocytes in the mammalian brain.

The role of Pten in the Hematopoietic System and the Hematopoietic Stem Cells

Résumé Identification, localisation et activation des cellules souches hématopoiétiques dormantes in vivo Les cellules souches somatiques sont présentes dans la majorité des tissus régénératifs comme la peau, l'épithélium intestinal et le système hématopoiétique. A partir d'une seule cellule, elles ont les capacités de produire d'autres cellules souches du même type (auto-renouvellement) et d'engendrer un ensemble défini de cellules progénitrices différenciées qui vont maintenir ou réparer leur tissu hôte. Les cellules souches adultes les mieux caractérisées sont les cellules souches hématopoiétiques (HSC), localisées dans la moelle osseuse. Un des buts de mon travail de doctorat était de caractériser plus en profondeur la localisation des HSCs endogènes in vivo. Pour ce faire, la technique "label retaining assay", se basant sur la division peu fréquentes et sur la dormance des cellules souches, a été utilisée. Après un marquage des souris avec du BrdU (analogue à l'ADN) suivi d'une longue période sans BrdU, les cellules ayant incorporés le marquage ("label retaining cells" LCRs) ont pu être identifiées dans la moelle osseuse. Ces cellules LCRs étaient enrichies 300 fois en cellules de phenotype HSC et, en utilisant de la cytofluorométrie, il a pu être montré qu'environ 15% de toutes les HSCs d'une souris restent dormantes durant plusieures semaines. Ces HSCs dormantes à long terme ne sont probablement pas impliquées dans la maintenance de 'hématopoièse. Par contre, on assiste à l'activation rapide de ces HSCs dormantes lors d'une blessure, comme une ablation myéloide. Elles re-entrent alors en cycle cellulaire et sont essentielles pour une génération rapide des cellules progénitrices et matures qui vont remplacer les cellules perdues. De plus, la détection des LCRs, combinée avec l'utilisation du marqueur de HSCs c-kit, peut être utilisée pour la localisation des HSCs dormantes présentes dans la paroi endostéale de la cavité osseuse. De manière surprenante, les LCRs c-kit+ ont surtout étés trouvées isolées en cellule unique, suggérant que le micro-environement spécifique entourant et maintenant les HSCs, appelé niche, pourrait être très réduit et abriter une seule HSC par niche. Rôles complexes du gène supresseur de tumeur Pten dans le système hématopoiétique La phosphatase PTEN disparaît dans certains cancers héréditaires ou sporadiques humains, comme les gliomes, les cancers de l'utérus ou du sein. Pten inhibe la voie de signalisation de la PI3-kinase et joue un rôle clé dans l'apoptose, la croissance, la prolifération et la migration cellulaire. Notre but était d'étudier le rôle de Pten dans les HSC normale et durant la formation de leucémies. Pour ce faire, nous avons généré un modèle murin dans lequel le gène Pten peut être supprimé dans les cellules hématopoiétiques, incluant les HSCs. Ceci a été possible en croissant l'allèle conditionnelle ptenflox soit avec le transgène MxCre inductible par l'interféron α soit avec le transgène Scl-CreERt inductible par le tamoxifen. Ceci permet la conversion de l'allèle ptenflox en l'allèle nul PtenΔ dans les HSCs et les autres types cellulaires hématopoiétiques. Les souris mutantes Pten développent une splénomégalie massive causée par une expansion dramatiques de toutes les cellules myéloides. De manière interessante, alors que le nombre de HSCs dans la moelle osseuse diminue progressivement, le nombre des HSCs dans la rate augmente de manière proportionnelle. Etrangement, les analyses de cycle cellulaire ont montrés que Pten n'avait que peu ou pas d'effet sur la dormance des HSCs ou sur leur autorenouvellement. En revanche, une augmentation massive du niveau de la cytokine de mobilisation G-CSF a été détéctée dans le serum sanguin, suggérant que la suppression de Pten stimulerait la mobilisation et la migration des HSC de la moelle osseuse vers la rate. Finallement, la transplantation de moelle osseuse délétée en Pten dans des souris immuno-déficientes montre que Pten fonctionnerait comme un suppresseur de tumeur dans le système hématopoiétique car son absence entraîne la formation rapide de leucémies lymphocytaires. Summary Identification, localization and activation of dormant hematopoietic stun cells in vivo Somatic stem cells are present in most self-renewing tissues including the skin, the intestinal epithelium and the hematopoietic system. On a single cell basis they have the capacity to produce more stem cells of the same phenotype (self-renewal) and to give rise to a defined set of mature differentiated progeny, responsible for the maintenance or repair of the host tissue. The best characterized adult stem cell is the hematopoietic stem cell (HSC) located in the bone marrow. One goal of my thesis work was to further characterize the location of endogenous HSCs in vivo. To do this, a technique called "label retaining assay» was used which takes advantage of the fact that stem cells (including HSCs) divide very infrequently and can be dormant for months. After labeling mice with the DNA analogue BrdU followed by a long BrdU free "chase", BrdU "label retaining cells" (CRCs) could be identified in the bone marrow. These CRCs were 300-fold enriched for phenotypic HSCs and by using flow cytometry analysis it could be shown that about 15% of all HSCs in the mouse are dormant for many weeks. Our results suggest that these long-term dormant HSCs are unlikely to be involved in homeostatic maintenance. However they are rapidly activated and reenter the cell cycle in response to injury signals such as myeloid ablation. In addition, detection of LRCs in combination with the HSC marker c-Kit could be used to locate engrafted dormant HSCs close to the endosteal lining of the bone marrow cavities. Most surprisingly, c-Kit+LRCs were found predominantly as single cells suggesting that the specific stem cell maintaining microenvironment, called niche, has limited space and may house only single HSCs. Complex roles of the tumor suppressor gene Pten in the hematopoietic system. The phosphatase PTEN is lost in hereditary and sporadic forms of human cancers, including gliomas, endometrial and breast cancers. Pten inhibits the PI3-kina.se pathway and plays a key role in apoptosis, cell growth, proliferation and migration. Our aim was to study the role of Pten in normal HSCs and during leukemia formation. To do this, we generated a mouse model in which the Pten gene can be deleted in hematopoietic cells including HSCs. This was achieved by crossing the conditional ptenflox allele with either the interferona inducible MxCre or the tamoxifen inducible Scl-CreERT transgene. This allowed the conversion of the ptenflox allele into a pterr' null allele in HSCs and other hematopoietic cell types. As a result Pten mutant mice developed massive splenomegaly due to a dramatic expansion of all myeloid cells. Interestingly, while the number of bone marrow HSCs progressively decreased, the number of HSCs in the spleen increased to a similar extent. Unexpectedly, extensive cell cycle analysis showed that Pten had little or no effect on HSC dormancy or HSC self-renewal. Instead, dramatically increased levels of the mobilizing cytokine G-CSF were detected in the blood serum suggesting that loss-of Pten stimulates mobilization and migration of HSC from the BM to the spleen. Finally, transplantation of Pten deficient BM cells into immuno-compromised mice showed that Pten can function as a tumor suppressor in the hematopoietic system and that its absence leads to the rapid formation of T cell leukemia.

Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human.

Ligands of the tumor necrosis factor superfamily (TNFSF) (4-1BBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA1, EDA2, FasL, GITRL, LIGHT, lymphotoxin alpha, lymphotoxin alphabeta, OX40L, RANKL, TL1A, TNF, TWEAK, and TRAIL) bind members of the TNF receptor superfamily (TNFRSF). A comprehensive survey of ligand-receptor interactions was performed using a flow cytometry-based assay. All ligands engaged between one and five receptors, whereas most receptors only bound one to three ligands. The receptors DR6, RELT, TROY, NGFR, and mouse TNFRH3 did not interact with any of the known TNFSF ligands, suggesting that they either bind other types of ligands, function in a ligand-independent manner, or bind ligands that remain to be identified. The study revealed that ligand-receptor pairs are either cross-reactive between human and mouse (e.g. Tweak/Fn14, RANK/RANKL), strictly species-specific (GITR/GITRL), or partially species-specific (e.g. OX40/OX40L, CD40/CD40L). Interestingly, the receptor binding patterns of lymphotoxin alpha and alphabeta are redundant in the human but not in the mouse system. Ligand oligomerization allowed detection of weak interactions, such as that of human TNF with mouse TNFR2. In addition, mouse APRIL exists as two different splice variants differing by a single amino acid. Although human APRIL does not interact with BAFF-R, the shorter variant of mouse APRIL exhibits weak but detectable binding to mouse BAFF-R.

Stable isotope geochemistry and formation mechanisms of quartz veins; Extreme paleoaltitudes of the Central Alps in the Neogene

Quartz veins ranging in size from less than 50 cm length and 5 cm width to greater than 10 m in length and 5 m in width are found throughout the Central Swiss Alps. In some cases, the veins are completely filled with milky quartz, while in others, sometimes spectacular void-filling quartz crystals are found. The style of vein filling and size is controlled by host rock composition and deformation history. Temperatures of vein formation, estimated using stable isotope thermometry and mineral equilibria, cover a range of 450 degrees C down to 150 degrees C. Vein formation started at 18 to 20 Ma and continued for over 10 My. The oxygen isotope values of quartz veins range from 10 to 20 permil, and in almost all cases are equal to those of the hosting lithology. The strongly rock-buffered veins imply a low fluid/rock ratio and minimal fluid flow. In order to explain massive, nearly morromineralic quartz formation without exceptionally large fluid fluxes, a mechanism of differential pressure and silica diffusion, combined with pressure solution, is proposed for early vein formation. Fluid inclusions and hydrous minerals in late-formed veins have extremely low delta D values, consistent with meteoric water infiltration. The change from rock-buffered, static fluid to infiltration from above can be explained in terms of changes in the large-scale deformation style occurring between 20 and 15 Ma. The rapid cooling of the Central Alps identified in previous studies may be explained in part, by infiltration of cold meteoric waters along fracture systems down to depths of 10 km or more. An average water flux of 0.15 cm 3 cm(-2)yr(-1) entering the rock and reemerging heated by 40 degrees C is sufficient to cool rock at 10 km depth by 100 degrees C in 5 million years. The very negative delta D values of < -130 permil for the late stage fluids are well below the annual average values measured in meteoric water in the region today. The low fossil delta D values indicate that the Central Alps were at a higher elevation in the Neogene. Such a conclusion is supported by an earlier work, where a paleoaltitude of 5000 meters was proposed on the basis of large erratic boulders found at low elevations far from their origin.

Nimesulide, a cyclooxygenase-2 preferential inhibitor, impairs renal function in the newborn rabbit.

Tocolysis with nonsteroidal anti-inflammatory drugs (NSAIDs) has been widely accepted for several years. Recently, the use of the cyclooxygenase-2 (COX2) preferential NSAID nimesulide has been proposed. However, data reporting neonatal acute renal failure or irreversible end-stage renal failure after maternal ingestion of nimesulide question the safety of this drug for the fetus and the neonate. Therefore, this study was designed to define the renal effects of nimesulide in newborn rabbits. Experiments were performed in 28 newborn rabbits. Renal function and hemodynamic parameters were measured using inulin and para-aminohippuric acid clearances as markers of GFR and renal blood flow, respectively. After a control period, nimesulide 2, 20, or 200 microg/kg was given as an i.v. bolus, followed by a 0.05, 0.5, or 5 microg.kg(-1).min(-1) infusion. Nimesulide administration induced a significant dose-dependent increase in renal vascular resistance (29, 37, and 92%, respectively), with a concomitant decrease in diuresis (-5, -23, and -44%), GFR (-12, -23, and -47%), and renal blood flow (-23, -23, and -48%). These results are in contrast with recent reports claiming that selective COX2 inhibition could be safer for the kidney than nonselective NSAIDs. These experiments confirm that prostaglandins, by maintaining renal vasodilation, play a key role in the delicate balance regulating neonatal GFR. We conclude that COX2-selective/preferential inhibitors thus should be prescribed with the same caution as nonselective NSAIDs during pregnancy and in the neonatal period.

Oligo-Miocene extensional tectonics and fluid flow across the Northern Snake Range detachment system, Nevada

The Northern Snake Range (Nevada) represents a spectacular example of a metamorphic core complex and exposes a complete section from the mylonitic footwall into the hanging wall of a fossil detachment system. Paired geochronological and stable isotopic data of mylonitic quartzite within the detachment footwall reveal that ductile deformation and infiltration of meteoric fluids occurred between 27 and 23 Ma. Ar-40/Ar-39 ages display complex recrystallization-cooling relationships but decrease systematically from 26.9 +/- 0.2 Ma at the top to 21.3 +/- 0.2 Ma at the bottom of footwall mylonite. Hydrogen isotope (delta D) values in white mica are very low (-150 to -145 %) within the top 80-90 m of detachment footwall, in contrast to values obtained from the deeper part of the section where values range from -77 to -64 %, suggesting that time-integrated interaction between rock and meteoric fluid was restricted to the uppermost part of the mylonitic footwall. Pervasive mica-water hydrogen isotope exchange is difficult to reconcile with models of Ar-40 loss during mylonitization solely by volume diffusion. Rather, we interpret the Ar-40/Ar-39 ages of white mica with low-delta D values to date syn-mylonitic hydrogen and argon isotope exchange, and we conclude that the hydrothermal system of the Northern Snake Range was active during late Oligocene (27-23 Ma) and has been exhumed by the combined effects of ductile strain, extensional detachment faulting, and erosion.

From the vendor in the hemp shop to the dealer in the alleyway? A comparison of the sources of supply of cannabis in a time of 'de-normalising' cannabis use.

Objectives: After several years of increasing 'normalisation' of cannabis use in Switzerland at the beginning of the new millennium, a reversed tendency, marked among others by a more stringent law-enforcement, set in. The presentation examines the question of where adolescents and young adults obtained cannabis, within the context of this societal change. In addition, it compares the sources of supply for cannabis with those found in studies of other European countries. Methods: Analyses are based on data from the Swiss Cannabis Monitoring Study. As part of this longitudinal, representative population survey, more than 5000 adolescents and young adults were interviewed by telephone on the topic of cannabis. Within the total sample, 593 (2004) or 554 (2007) respectively, current cannabis users replied to the questions on sources of supply. Changes in law-enforcement and societal climate concerning cannabis are assessed based on relevant literature, media reports and parliamentary discussions. Results: Whereas 22% of cannabis users stated in 2004 that they bought their cannabis from vendors in hemp shops, this proportion drastically decreased to 6% three years later. At the same time, cannabis was obtained increasingly from friends, while the proportion of users who purchased cannabis from dealers in the alleyway, more than doubled from 6% (2004) to 13% (2007). It was male cannabis users, and in particular, young adult and frequent users, who have moved into the alleyways. Generally, users who buy cannabis in the alleyway show more cannabis-related problems than those who mainly name other sources of supply, even when adjusted for sex, age and frequency of cannabis use. Discussion: Possible consequences of these changes in cannabis supply, like the risk of merging a previously cannabis-only market with other 'harder' drugs markets, are discussed.

Artifacts in the analysis of thioridazine and other neuroleptics.

Although the sensitivity to light of thioridazine and its metabolites has been described, the problem does not seem to be widely acknowledged. Indeed, a survey of the literature shows that assays of these compounds under light-protected conditions have been performed only in a few of the numerous analytical studies on this drug. In the present study, thioridazine, its metabolites, and 18 other neuroleptics were tested for their sensitivity to light under conditions used for their analysis. The results show that light significantly affects the analysis of thioridazine and its metabolites. It readily causes the racemization of the isomeric pairs of thioridazine 5-sulphoxide and greatly decreases the concentration of thioridazine. This sensitivity to light varied with the medium used (most sensitive in acidic media) and also with the molecule (in order of decreasing sensitivity: thioridazine > mesoridazine > sulforidazine). Degradation in neutral or basic media was slow, with the exception of mesoridazine in a neutral medium. Twelve other phenothiazines tested, as well as chlorprotixene, a thioxanthene drug, were found to be sensitive to light in acidic media, whereas flupenthixol and zuclopenthixol (two thioxanthenes), clozapine, fluperlapine, and haloperidol (a butyrophenone) did not seem to be affected. In addition to being sensitive to light, some compounds may be readily oxidized by peroxide-containing solvents.

Identification of early metabolic markers of various degrees of ischemia in the mouse brain by localized H-1 magnetic resonance spectroscopy

Objectives: Magnetic resonance (MR) imaging and spectroscopy (MRS) allow the establishment of the anatomical evolution and neurochemical profiles of ischemic lesions. The aim of the present study was to identify markers of reversible and irreversible damage by comparing the effects of 10-mins middle cerebral artery occlusion (MCAO), mimicking a transient ischemic attack, with the effects of 30-mins MCAO, inducing a striatal lesion. Methods: ICR-CD1 mice were subjected to 10-mins (n = 11) or 30-mins (n = 9) endoluminal MCAO by filament technique at 0 h. The regional cerebral blood flow (CBF) was monitored in all animals by laser- Doppler flowmetry with a flexible probe fixed on the skull with < 20% of baseline CBF during ischemia and > 70% during reperfusion. All MR studies were carried out in a horizontal 14.1T magnet. Fast spin echo images with T2-weighted parameters were acquired to localize the volume of interest and evaluate the lesion size. Immediately after adjustment of field inhomogeneities, localized 1H MRS was applied to obtain the neurochemical profile from the striatum (6 to 8 microliters). Six animals (sham group) underwent nearly identical procedures without MCAO. Results: The 10-mins MCAO induced no MR- or histologically detectable lesion in most of the mice and a small lesion in some of them. We thus had two groups with the same duration of ischemia but a different outcome, which could be compared to sham-operated mice and more severe ischemic mice (30-mins MCAO). Lactate increase, a hallmark of ischemic insult, was only detected significantly after 30-mins MCAO, whereas at 3 h post ischemia, glutamine was increased in all ischemic mice independently of duration and outcome. In contrast, glutamate, and even more so, N-acetyl-aspartate, decreased only in those mice exhibiting visible lesions on T2-weighted images at 24 h. Conclusions: These results suggest that an increased glutamine/glutamate ratio is a sensitive marker indicating the presence of an excitotoxic insult. Glutamate and NAA, on the other hand, appear to predict permanent neuronal damage. In conclusion, as early as 3 h post ischemia, it is possible to identify early metabolic markers manifesting the presence of a mild ischemic insult as well as the lesion outcome at 24 h.

Calculation of correction factors for ionization chamber measurements with small fields in low-density media.

The quantity of interest for high-energy photon beam therapy recommended by most dosimetric protocols is the absorbed dose to water. Thus, ionization chambers are calibrated in absorbed dose to water, which is the same quantity as what is calculated by most treatment planning systems (TPS). However, when measurements are performed in a low-density medium, the presence of the ionization chamber generates a perturbation at the level of the secondary particle range. Therefore, the measured quantity is close to the absorbed dose to a volume of water equivalent to the chamber volume. This quantity is not equivalent to the dose calculated by a TPS, which is the absorbed dose to an infinitesimally small volume of water. This phenomenon can lead to an overestimation of the absorbed dose measured with an ionization chamber of up to 40% in extreme cases. In this paper, we propose a method to calculate correction factors based on the Monte Carlo simulations. These correction factors are obtained by the ratio of the absorbed dose to water in a low-density medium □D(w,Q,V1)(low) averaged over a scoring volume V₁ for a geometry where V₁ is filled with the low-density medium and the absorbed dose to water □D(w,QV2)(low) averaged over a volume V₂ for a geometry where V₂ is filled with water. In the Monte Carlo simulations, □D(w,QV2)(low) is obtained by replacing the volume of the ionization chamber by an equivalent volume of water, according to the definition of the absorbed dose to water. The method is validated in two different configurations which allowed us to study the behavior of this correction factor as a function of depth in phantom, photon beam energy, phantom density and field size.

MOLECULAR REORGANIZATION FOLLOWING SENSORY STIMULATION IN THE ADULT MOUSE BARREL CORTEX

Sensory information is an important factor in shaping neuronal circuits during development and adulthood. In the barrel cortex of adult rodents, cells from layer IV are able to adapt their functional state to an increased flow of sensory information from the mystacial whisker follicles. Previous studies in our group have shown that whisker stimulation induces the formation of inhibitory synapses in the corresponding barrel (Knott et al., 2002) and decreases neuronal responses toward the deflection of the stimulated whisker (Quairiaux et al., 2007). Together these observations have turned the barrel cortex into a model to study homeostatic plasticity. At the cellular level, neuronal activity triggers intracellular signaling cascades leading to a transcriptional response. To further characterize the molecular pathways involved in the synaptic changes after whisker stimulation in the adult mouse, a previous doctoral student in our group performed a microarray analysis on laser-dissected barrels in sections through layer IV. This study identified the regulation (up and down) of a series of genes in the stimulated barrels (thesis of Johnston-Wenger, 2010). We here focused on ten genes that presented the highest fold change according to the microarray analysis. Out of these genes, 7 are known as neuronal activity-dependent genes (Tnncl, Nptx2, Sorcs3, Ptgs2, Nr4a2, Npas4 and Adcyapl) whereas three have so far not been related to neuronal plasticity (Scn7a, Pcdhl5 and Cede3). The study aimed at confirming the results of the microarray analysis and localizing molecular modifications in the stimulated barrel column at the cellular level. In situ hybridization for Pcdhl5 after different periods of whisker stimulation (3, 6, 9, 15, 24 hrs) allowed us to confirm that the 1.25 fold change used for the microarray analysis is an appropriate threshold for considering a regulation significant after sensory-stimulation. Moreover, we confirmed with in situ hybridization a significant upregulation of the genes of interest in the stimulated barrels. In situ hybridization and immunohistochemistry allowed us to observe the distribution of the genes of interest and the corresponding protein products at the cellular level. Three observations were made: 1) alterations of the expression was restricted to the stimulated barrels for all genes tested; 2) within a barrel column not all cells responded to whisker stimulation with an altered gene expression; 3) in the stimulated barrels, two different patterns of mRNA and protein expression can be distinguished. We hypothesize that this segregation of the activity-induced gene expression reflects the segregation of the two principal thalamocortical pathways conveying the sensory information to the barrel cortex. Moreover, only neurons reaching the critical threshold will modify their gene expression program resulting in structural as well as physiological modifications that prevent the subsequent propagation of the excess of excitation to the postsynaptic targets. The activity-induced gene expression is therefore adapted in a cell-type-specific manner to induce a homeostatic response to the entire neuronal network involved in the integration of the sensory information. This to our knowledge the first study showing the distinct, but complementary contribution of the two thalamocortical pathways in experience-dependent plasticity in the adult mouse barrel cortex. -- L'information sensorielle nous permet de continuellement façonner nos circuits neuronaux autant durant le développement qu'à l'âge adulte. Chez le rongeur l'information sensorielle perçue par les vibrisses est intégrée au niveau du cortex somatosensoriel primaire (appelé en anglais « barrel cortex ») dont les cellules de la couche IV sont capables d'adapter leur état fonctionnel en réponse à une augmentation d'activité neuronale. Ce modèle expérimental a permis à notre groupe de recherche d'observer des changements rapides du circuit neuronal en fonction de l'activité sensorielle. En effet, la stimulation continue d'une vibrisse d'une souris adulte pendant 24 heures induit non seulement un remaniement synaptique (Knott et al., 2002), mais également des changements physiologiques au niveau des neurones du tonneau correspondant (Quairiaux et al., 2007). Ces observations nous permettent d'affirmer que le « barrel cortex » est un modèle approprié pour y étudier la plasticité synaptique. Au niveau cellulaire, l'activité neuronale déclenche des cascades de signalisation intracellulaire résultant en une réponse transcriptionnelle. Afin de caractériser les voies moléculaires impliquées dans la plasticité synaptique, une puce à ARN nous a permis de comparer l'expression de gènes entre un tonneau correspondant à une vibrisse stimulée et un tonneau d'une vibrisse non-stimulée (Nathalie). Cette analyse a révélé un certain nombre de gènes régulés de manière positive ou négative par l'augmentation de l'activité neuronale. Nous nous sommes concentrés sur 10 gènes dont l'expression est fortement régulée. L'expression de sept d'entre eux a déjà été démontrée comme dépendante de l'activité neuronale (Tnncl, Nptx2, Sorcs3, Ptgs2, Nr4a2, Npas4 otAdcyapl) alors que l'expression des trois autres (Scn7a, Pcdhl5 et Cedei) n'a pour le moment pas encore été liée à la plasticité neuronale. Le but de cette thèse est de confirmer les résultats de la puce à ARN et de déterminer dans quel type cellulaire ces gènes sont exprimés. L'hybridation in situ pour le gène Pcdhl5, après différentes périodes de stimulation des vibrisses (3, 6, 9, 15 et 24 heures), nous a permis de confirmer que le seuil de 1.25x utilisé dans l'analyse de la puce à ARN est approprié pour considérer qu'un gène est régulé de manière significative par la stimulation sensorielle. Nous avons également pu confirmer à l'aide de cette technique que la stimulation sensorielle augmente significativement l'expression de ces dix gènes. L'expression de ces gènes au niveau cellulaire a été observée à l'aide des techniques d'hybridation in situ et d'immunohistochimie. Trois observations ont été faites : 1) la régulation de ces gènes est restreinte aux tonneaux correspondants aux vibrisses stimulées ; 2) au niveau d'une colonne corticale correspondant aux vibrisses stimulées, seules certaines cellules présentent une altération de leur expression génique ; 3) au niveau des tonneaux stimulés, deux profils d'expression d'ARNm et de protéines sont observés. Notre hypothèse est que cette distribution pourrait correspondre à la terminaison ségrégée des deux voies thalamocortical qui amènent l'information sensorielle dans le cortex cérébral. De plus, seul les neurones atteignant le seuil critique d'activation modifient leur expression génique en réponse à la stimulation sensorielle. Ces changements d'expression géniques vont permettre à la cellule de modifier ses propriétés structurales et physiologiques de manière a prevenir la propagation d'un excès d'activité neuronale au niveau de ses cibles postsynaptics. L'activité neuronale agit donc spécifiquement sur certains types cellulaires de maniere a induire une réponse homéostatique au niveau du réseau neuronal impliqué dans l'integration de l'information sensorielle. Nos travaux démontrent pour une première fois que les deux voies sensorielles contribuent d'une manière distincte et complémentaire à la plasticité corticale induite par un changement de l'activité sensorielle chez la souris adulte.

SNARE protein expression in synaptic terminals and astrocytes in the adult hippocampus: a comparative analysis.

Several evidences suggest that astrocytes release small transmitter molecules, peptides, and protein factors via regulated exocytosis, implying that they function as specialized neurosecretory cells. However, very little is known about the molecular and functional properties of regulated secretion in astrocytes in the adult brain. Establishing these properties is central to the understanding of the communication mode(s) of these cells and their role(s) in the control of synaptic functions and of cerebral blood flow. In this study, we have set-up a high-resolution confocal microscopy approach to distinguish protein expression in astrocytic structures and neighboring synaptic terminals in adult brain tissue. This approach was applied to investigate the expression pattern of core SNARE proteins for vesicle fusion in the dentate gyrus and CA1 regions of the mouse hippocampus. Our comparative analysis shows that astrocytes abundantly express, in their cell body and main processes, all three protein partners necessary to form an operational SNARE complex but not in the same isoforms expressed in neighbouring synaptic terminals. Thus, SNAP25 and VAMP2 are absent from astrocytic processes and typically concentrated in terminals, while SNAP23 and VAMP3 have the opposite expression pattern. Syntaxin 1 is present in both synaptic terminals and astrocytes. These data support the view that astrocytes in the adult hippocampus can communicate via regulated exocytosis and also indicates that astrocytic exocytosis may differ in its properties from action potential-dependent exocytosis at neuronal synapses, as it relies on a distinctive set of SNARE proteins.

The arbuscular mycorrhizal fungus Glomus intraradices is haploid and has a small genome size in the lower limit of eukaryotes.

The genome size, complexity, and ploidy of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was determined using flow cytometry, reassociation kinetics, and genomic reconstruction. Nuclei of G. intraradices from in vitro culture, were analyzed by flow cytometry. The estimated average length of DNA per nucleus was 14.07+/-3.52 Mb. Reassociation kinetics on G. intraradices DNA indicated a haploid genome size of approximately 16.54 Mb, comprising 88.36% single copy DNA, 1.59% repetitive DNA, and 10.05% fold-back DNA. To determine ploidy, the DNA content per nucleus measured by flow cytometry was compared with the genome estimate of reassociation kinetics. G. intraradices was found to have a DNA index (DNA per nucleus per haploid genome size) of approximately 0.9, indicating that it is haploid. Genomic DNA of G. intraradices was also analyzed by genomic reconstruction using four genes (Malate synthase, RecA, Rad32, and Hsp88). Because we used flow cytometry and reassociation kinetics to reveal the genome size of G. intraradices and show that it is haploid, then a similar value for genome size should be found when using genomic reconstruction as long as the genes studied are single copy. The average genome size estimate was 15.74+/-1.69 Mb indicating that these four genes are single copy per haploid genome and per nucleus of G. intraradices. Our results show that the genome size of G. intraradices is much smaller than estimates of other AMF and that the unusually high within-spore genetic variation that is seen in this fungus cannot be due to high ploidy.

Genetic clusters and sex-biased gene flow in a unicolonial Formica ant.

BACKGROUND: Animal societies are diverse, ranging from small family-based groups to extraordinarily large social networks in which many unrelated individuals interact. At the extreme of this continuum, some ant species form unicolonial populations in which workers and queens can move among multiple interconnected nests without eliciting aggression. Although unicoloniality has been mostly studied in invasive ants, it also occurs in some native non-invasive species. Unicoloniality is commonly associated with very high queen number, which may result in levels of relatedness among nestmates being so low as to raise the question of the maintenance of altruism by kin selection in such systems. However, the actual relatedness among cooperating individuals critically depends on effective dispersal and the ensuing pattern of genetic structuring. In order to better understand the evolution of unicoloniality in native non-invasive ants, we investigated the fine-scale population genetic structure and gene flow in three unicolonial populations of the wood ant F. paralugubris. RESULTS: The analysis of geo-referenced microsatellite genotypes and mitochondrial haplotypes revealed the presence of cryptic clusters of genetically-differentiated nests in the three populations of F. paralugubris. Because of this spatial genetic heterogeneity, members of the same clusters were moderately but significantly related. The comparison of nuclear (microsatellite) and mitochondrial differentiation indicated that effective gene flow was male-biased in all populations. CONCLUSION: The three unicolonial populations exhibited male-biased and mostly local gene flow. The high number of queens per nest, exchanges among neighbouring nests and restricted long-distance gene flow resulted in large clusters of genetically similar nests. The positive relatedness among clustermates suggests that kin selection may still contribute to the maintenance of altruism in unicolonial populations if competition occurs among clusters.