Lensemble du travail de recherche réalisé s'est concentré sur l'évaluation du rôle fonctionnel que peut exercer le gène circadien Per2 sur les capacités de prolifération et différenciation des cellules souches/progénitrices à l'origine de la neurogenèse hippocampique. Ce travail a comporté d'une part, une cartographie phénotypique exhaustive de l'identité des cellules exprimant la protéine PER2 au sein de la structure hippocampique, et d'autre part une étude approfondie des conséquences de la l'invalidation de ce gène sur la régulation de la neurogenèse dans l'hippocampe de souris adultes. Dans la première partie de notre travail, nous avons démontré par une analyse immunohistochimique détaillée, qu'au niveau du gyrus dentelé (DG) de souris adultes, les cellules proliférantes exprimaient la protéine PER2 et que cette expression persistait dans les cellules de la lignée neuronale à différents stades de maturation. Par ailleurs, à l'inverse du noyau suprachiasmatique (centre générateur des rythmes circadiens), nous avons également pu observer une expression constante de cette protéine durant une période de 24h (Borgs et al, soumis). Dans la seconde partie de notre travail, nous nous sommes interrrogés sur le rôle fonctionnel que pouvait exercer le facteur de transcription circadien Per2 dans le DG de souris adultes. Nous avons montré que linvalidation de ce gène entraine dans le DG des souris déficientes pour la protéine PER2, une augmentation significative de la prolifération des progéntieurs neuronaux, ainsi que du nombre de neurones immatures. Cependant, nous navons observé aucune différence dans la génération de neurones matures (neurogenèse) entre le DG de souris sauvages et de souris invalidées pour Per2. Nos données ont révélé que le surplus de cellules en prolifération et de neurones immatures observés dans le DG de souris délétées pour Per2 apparaît donc totalement compensé par une augmentation de la mort cellulaire (Borgs et al, soumis). Pour étudier limplication fonctionnel de la protéine PER2 sur le contrôl de la prolifération de progéniteurs/cellules souches à lorigine de la neurogenèse adulte, nous avons mis au point la culture en suspension de cellules souches/progénitrices issues du DG post-natale de souris sauvages et déficientes pour Per2. Après 5 jours de culture, nous avons observé la formation de neurosphères dont la taille et dont la croissance était plus importante chez les souris déficientes pour Per2 que chez leurs homologues sauvages. Ce modèle de culture de DG nous a permis détudier de façon plus présice le destin cellulaire emprunté par les cellules proliférantes/souches dans le modèle muté, comparé au modèle sauvage. En condition de culture favorisant la différenciation, nous avons observé un plus grand nombre de neurones générés à partir des neurosphères issues de cellules de DG de souris mutées pour PER2. Ce modèle de culture de cellules progénitrices/souches issues du DG, confirme les résultats précédemment obtenus concernant le rôle de Per2 dans le contrôle de la prolifération et de la génération de nouveaux neurones in vivo. Parallèlement, nous avons tenté de déterminer si lexpression de Per2 pouvait exercer un rôle similaire au DG au sein de la zone sous ventriculaire antérieure (SVZ), la seconde zone où persiste de la neurogenèse tout au long de la vie. La SVZ du cerveau adulte représente un réservoir de progéniteurs proliférant qui vont cheminer le long dun courant rostral de migration pour atteindre le bulbe olfactif dans lequel ils vont se différencier en neurones. La protéine Per2 se révèle être exprimée dans les progéniteurs en prolifération exprimant Ki67. Tout comme dans le DG de souris adultes déficientes pour Per2, nous avons dénombré in vivo et in vitro une augmentation importante du nombre de cellules en prolifération comparé aux souris sauvages. Per2 semble donc être un des protagonistes impliqué dans la régulation de la prolifération et de la différenciation des progéniteurs/cellules souches à lorigine de la neurogenèse hippocampique.

Background: Ischemic stroke ranks as the third major cause of clinical mortality and the leading cause of handicap in adults. Each year, stroke occurs in about 30,000 Swedes. The severity of an acute ischemic stroke depends mainly on the degree and duration of local cerebral blood flow (lCBF) reduction. Prompt reperfusion improves neurological deficits, spontaneous electrical activity, energy metabolism, cerebral protein synthesis (CPS), and tissue repair, among which cell proliferation (neurogenesis, gliosis) and revascularization (angiogenesis) may have important functional and therapeutic implications. Aims of the thesis: (1) To establish the photothrombotic ring stroke(PRS) model with late spontaneous reperfusion in adult mice; (2) To explore angiogenesis and neurogenesis in adult brain after focal cerebral ischemia. Materials and Methods: The PRS model in C57 BL adult mice and the middle cerebral artery suture occlusion (MCAO) model in adult Wistar rats were used. The 5-bromodeoxyuridine (BrdU) was delivered into animal after stroke induction to label DNA duplication. CBF, CPS and adenosine triphosphate (ATP) were measured by laser-Doppler flowmetry (LDF), [14C]–Iodoantipyrine and [3H]-Leucine double tracer autoradiography, and bioluminescence, respectively. Immunocytochemistry / immunofluoresence were performed to detect different proteins. The cell marker colocalization was analyzed by three-dimension (3-D) confocal. The cell counting was performed with a stereological counting system. Results: The PRS model was established in adult mice by irradiating the exposed skull with a 514.5 nm argon laser ring beam (3 mm diameter, 0.21 mm thick) at an intensity of 0.65 W/cm2 for 60s, with concurrent erythrosin B (4.25 mg/kg) intravenous infusion for 15s. The central cortical region within the ring locus was progressively encroached by an annular ring-shaped perfusion deficit, where lCBF LDF declined promptly to 43% of the baseline value at 30 min post irradiation. The lCBF-IAP amounted to 46-17-58 ml/100g/min, where CPS varied from 57-38-112% at 4h-48h-7days post ischemia. ATP declined at 4h, achieved its maximum level at 48h and was markedly reduced at 7 days postischemia. Morphologically, at 4h some neurons in the region at-risk appeared swollen, at 48h the majority were severely swollen, eosinophilic and pyknotic. Tissue morphology became partly restored at 7 days post stroke, when numerous cortical cells were immunolabeled by BrdU or the mitosis-specific marker phosphorylated histone H3 (Phos-H3). Some of these cells were even doubly immunopositive to the neuron-specific marker Neu N and the astrocyte marker GFAP, as analyzed by 3-D confocal. In adult rats exposed to MCAO, widespread BrdU-immunolabeled cells appeared in the cortex, ipsilateral striatum and dentate gyrus of the hippocampus. Some of which were doubleimmunolabeled by the neuron specific markers Map-2, β-tubulin III and Neu N as analyzed by 3-D confocal. As early as 24h postischemia, BrdU-immunopositive endothelial cells were aligned as microvessels, some of which exhibited distinguishable lumens in the ischemic boundary zone, where VEGF-A, B, C proteins and their receptors flt-1, fik-1, flt-4 were overexpressed at 72h after MCAO. Conclusion: PRS model in adult mice elicits a dynamic deterioration and then restoration of local CBF, CPS, ATP and tissue morphology in the spontaneously reperfused cerebral cortex at 7d after stroke, where cortical neurogenesis and gliosis occurred. In adult rats with MCAO, neurogenesis occurred at 30 and 60d in the penumbral cortex and striatum. Angiogenesis occurred as early as 24h, which contributed to the spontaneous reperfusion frequently observed in this setting of acute ischemic stroke.

Previous research on the relationship between spatial memory and adult hippocampal neurogenesis has been controversial. In the present study, neurogenesis was compared between two natural populations of the same species that differ in their reliance on spatial memory to cache and retrieve stored food. Western red squirrels store food in a single site whereas eastern red squirrels store food in multiple sites. Neurogenesis was assessed using endogenous markers of the number of proliferating cells (Ki-67) and the number of immature neurons (DCX), and neuronal recruitment was determined by measuring the area of the dentate gyrus of the hippocampus. The number of proliferating cells, immature neurons and neuronal recruitment were enhanced in the eastern compared to the western red squirrels, reflecting the food storing strategies used by the squirrels. This suggests that there is a positive correlation between adult hippocampal neurogenesis and spatial learning and memory.

Previous research on the relationship between spatial memory and adult hippocampal neurogenesis has been controversial. In the present study, neurogenesis was compared between two natural populations of the same species that differ in their reliance on spatial memory to cache and retrieve stored food. Western red squirrels store food in a single site whereas eastern red squirrels store food in multiple sites. Neurogenesis was assessed using endogenous markers of the number of proliferating cells (Ki-67) and the number of immature neurons (DCX), and neuronal recruitment was determined by measuring the area of the dentate gyrus of the hippocampus. The number of proliferating cells, immature neurons and neuronal recruitment were enhanced in the eastern compared to the western red squirrels, reflecting the food storing strategies used by the squirrels. This suggests that there is a positive correlation between adult hippocampal neurogenesis and spatial learning and memory.

Neurons, astrocytes and oligodendrocytes of the central nervous system (CNS) arise from a common pool of multipotent neuroepithelial progenitor cells lining the walls of the neural tube. Initially, neuroepithelial cells undergo symmetric proliferative divisions, thereby expanding the progenitor pool and determining the size of brain compartments. At the onset of neurogenesis, a subset of progenitors switch to asymmetric or terminal symmetric neurogenic divisions. Maintenance of progenitor cell population throughout the period of neurogenesis is essential to generate the full diversity of neuronal cell types and proper histological pattern. However, the mechanisms responsible for the maintenance of progenitor cells proliferation are far from being fully understood. The family of Y-box proteins is involved in control of proliferation and transformation in various normal and pathological cellular systems, and therefore was considered as a candidate to exert such a function. Y-box proteins have a capacity to bind DNA and RNA, thereby controlling gene expression from transcription to translation. This study aimed to examine the expression of mouse Y-box protein 3 (MSY3) in the developing nervous system and elucidate its putative role in regulation of proliferation of progenitor cells. As presented in this work, the MSY3 protein in the embryonic CNS is expressed solely in progenitor cells and not neurons. Moreover, as shown by two independent approaches: morphologically, i.e. using immunofluorescence and confocal microscopy, and biochemically, MSY3 expression is downregulated concomitantly with the spatiotemporal progression of neurogenesis. Interestingly, in preliminary results it was shown that MSY3 is expressed in Dcx-positive transient amplifying precursors in germinal zones of the adult brain, and in EGF-dependent neurospheres. To evaluate whether MSY3 could regulate the neurogenesis, the levels of the MSY3 protein in the progenitors were acutely downregulated or elevated by electroporation of RNAi or MSY3 expression plasmids, respectively. Neither premature reduction of MSY3 in the neuroepithelium (E9.5-E11.5) nor prolonged expression at the developmental stage when this protein is endogenously downregulated (E10.5-14.5) did affect proliferation versus the cell cycle exit of progenitors. Moreover, in Notch1-deficient progenitors in the cerebellar anlage, which exhibit precocious differentiation, MSY3 was not prematurely downregulated, suggesting that MSY3 also is not an early marker of differentiation. Differential centrifugation, immunoprecipitation and polysomal analysis performed in this study revealed that the MSY3 protein in the developing embryo, as well as in Neuro-2A cells, is associated with RNA. On a sucrose density gradient MSY3 co-fractionates with ribosomes and actively translating polysomes, suggesting that it might have a role in regulation of translation. However, downregulation or overexpression of MSY3 in the Neuro-2A cell line did not affect global translation rates. Other researchers suggested that the MSY3 protein has the redundant function with Y-box protein 1 (YB-1). Accordingly, in our system the MSY3 protein could be co-immunoprecipitated with YB-1. Importantly, developmentally regulated expression of MSY3 is not a hallmark of general translation apparatus, as several other proteins involved in translation did not show similar downregulation. To summarise, this work showed that the MSY3 protein is a marker of proliferation of progenitor cells in the embryonic and adult brain, being absent from neurons. Discovery of the molecular mechanism by which MSY3 exerts its role in the cell could provide the link between the translational machinery and proliferation.

During development of the nervous system, formation of specific connections between nerve cells depends on the stability of growing axons to reach appropriate target cells and form synapses. In culture, hippocampal neurons form numerous synapses by developing axonal and dendritic extensions. To elucidate principles of neuronal signaling and network establishment, creation of neuronal networks in which connectivity and pathways can be experimentally controlled is of great interest. In the present study we used a microcontact printing technique to control and study neurite outgrowth of hippocampal neurons in vitro. My preliminary results show that hippocampal neurons follow the microcontact printed pattern of poly-D-lysine (PDL). In doing so, neurons retain their morphology with normal subcellular distribution of various cell adhesion and synaptic molecules. However, the distribution of various axonal or dendrite components is altered. Hence we have developed a system in which isolated axons and dendrites align with inputs from very few neurons. With this technique we intend to study axon-dendrite communications on a spatially restricted and defined substrate.

Pastaruoju metu tyrimams dažniausiai naudojami žinduoliai, kadangi rezultatai šiek tiek atspindi žmogaus organizme vykstančius procesus. Šis darbas parengtas analizuojant besivystančias paukščio embriono sistemas: tai būdas, leidžiantis sutaupyti lėšų bei dėka trumpo embrioninio vystymosi laikotarpio suteikiantis galimybę operatyviam rezultatų gavimui. Iki šiol literatūroje neradome duomenų apie pirmojo raumens susitraukimo ypatybes, o šį procesą įtakojančios nervų sistemos vystymąsi apibūdina keletas hipotezių. Taigi darbo tikslas buvo išsiaiškinti pirmojo susitraukiančiojo raumens atsiradimo laiką, jo morfologiją (1) ir išanalizuoti ankstyvajame embriogenezės laikotarpyje kontraktilų aparatą įtakojančios nervų sistemos vystymosi ypatumus (2). Tyrimams naudojome kalakuto ir vištos embrionus – kadangi skirtingų porūšių skirtumai mums nebuvo svarbūs, palyginimas tarp minėtų paukščių embrionų neatliktas. Embrionai buvo stebimi in vivo, tyrimams in vitro atlikti buvo naudojami šie molekulinės biologijos metodai: paruoštų pjūvių dažymas histo- ir imunohistochemiškai, polimerazės grandininės reakcijos, ląstelių kultūrų auginimas ir Western blotas. Pirmasis aktyvus raumens susitraukimas kalakuto embrione atsiranda 19-22 Hamburgerio Hamiltono stadijoje, t.y., praėjus 116 val. nuo inkubacijos pradžios: porinis raumuo sudarytas iš keturių susiliejusių miomerų, yra 1 mm ilgio ir 0,1 mm pločio – šis iš dviejų ląstelių tipų sudarytas audinys atsakingas už pirmuosius aktyvius embriono... [toliau žr. visą tekstą] / The purpose of the study was to characterize the first contraction of an isolated muscle in turkey embryo and to determine whether at early stages of chicken brain development during neurogenesis, cells from the astrocytic lineage are present in relevant amounts, where they are located in the neural tube, and to what extend brain regional differences exist. MATERIAL AND METHODS. From the 3rd day of incubation on until the 6th day the embryos were continuously watched through a cellophane window in the eggshell. For histology the embryos were fixed in Bouin's fluid, then completely cut in serial sections of 5 microm thickness and stained according to Masson-Goldner's trichrome procedure plus resorcin-fuchsin. For gene expression analysis markers for stem cells, neurons and astrocytes were used. Immunohistochemistry was made against SMI 312 and GFAP antibodies and Western blot against GFAP as well. RESULTS. A paired muscle 1 mm long and 0.1 mm broad, derived by fusion of the four occipital myomeres, is responsible for the first individual contraction. The contraction produces a stretching in the neck region. The muscle named M. occipitalis primordialis consists of four end-to-end connected groups of mononucleated muscle cells. The muscle contains two types of cells according to the cell nuclei. The elastic rod-shaped notochord represents an endoskeleton. Immediately after contraction it brings the body of the embryo back into its former shape. We also demonstrate that specific... [to full text]

Deep brain stimulation (DBS) is currently being investigated as a therapy for treatment-resistant depression, with promising results. However, it is not clear whether or not DBS works via the same mechanisms as those induced by antidepressant medications. Processes currently implicated in antidepressant effects include neuroplastic changes and promotion of neurogenesis. We investigated the effects of chronic treatment with three different classes of antidepressants and DBS on markers of neuroplasticity (brain-derived neurotrophic factor, (BDNF), and phosphorylated cyclic-AMP regulatory element binding protein, (pCREB)) and neurogenesis (Ki-67, bromodeoxyuridine (BrdU) and doublecortin) in the rat hippocampus. No clear treatment effects were seen on BDNF, pCREB and Ki-67 levels. However all treatments caused increased levels of BrdU (range: 46%-96%) and doublecortin (8%-61%), although these effects were statistically significant only for DBS and amitriptyline, respectively. This overall pattern of results may suggest that diverse antidepressant treatments could possibly share common mechanisms involving cell survival and neuronal differentiation. Potentiated effects of DBS on cell survival may underlie its efficacy in treatment-resistant depression.

Deep brain stimulation (DBS) is currently being investigated as a therapy for treatment-resistant depression, with promising results. However, it is not clear whether or not DBS works via the same mechanisms as those induced by antidepressant medications. Processes currently implicated in antidepressant effects include neuroplastic changes and promotion of neurogenesis. We investigated the effects of chronic treatment with three different classes of antidepressants and DBS on markers of neuroplasticity (brain-derived neurotrophic factor, (BDNF), and phosphorylated cyclic-AMP regulatory element binding protein, (pCREB)) and neurogenesis (Ki-67, bromodeoxyuridine (BrdU) and doublecortin) in the rat hippocampus. No clear treatment effects were seen on BDNF, pCREB and Ki-67 levels. However all treatments caused increased levels of BrdU (range: 46%-96%) and doublecortin (8%-61%), although these effects were statistically significant only for DBS and amitriptyline, respectively. This overall pattern of results may suggest that diverse antidepressant treatments could possibly share common mechanisms involving cell survival and neuronal differentiation. Potentiated effects of DBS on cell survival may underlie its efficacy in treatment-resistant depression.

Adult hippocampal neurogenesis is regulated at various levels and by various factors. Genetic influence is an important key determinant of adult neurogenesis and exerts its effects at all levels. In vivo studies have suggested that adult hippocampal neurogenesis is highly variable and heritable among different laboratory strains of mice. To dissect the genetic effect from other contributing factors, it is necessary to study adult neurogenesis under highly controlled environment conditions. We extracted adult hippocampal precursor cells (AHPCs) from 20 strains of the BXD set of recombinant inbred mice, cultured them and studied the effect of genetic background on neurogenesis. The BXD panel consists of mouse lines derived from an intercross between inbred parentals C57BL/6J and DBA/2J. Both of the parentals are fully sequenced and all the strains are well characterized in terms of genotypic and phenotypic characteristics. This allows us to use advanced genetic techniques to identify novel genomic loci and gene-gene interactions important in adult neurogenesis. Comparison of the AHPCs from 20 BXD strains, with respect to cell proliferation and neuronal and astrocytic differentiation in vitro, revealed a large variation for these traits across the strains. Proliferation, as measured by BrdU incorporation, showed over two- fold differences between the extremes. Similar differences were observed for neurogenic (4-fold) and astrogenic differentiation (2-fold). These three traits all showed strong heritability values indicating that the differences were mainly attributed to the genetic component. QTL mapping, with these phenotypic data, revealed that there was no major contribution from single loci controlling these traits. Instead, we found many loci with smaller effects associated with these traits. Gene expression profiling using RNA samples from proliferating cultures of the 20 BXD mice strains yielded two cis eQTL candidates that directly regulated proliferation, LRP6 and Chchd8. LRP6 is well known as a co-receptor of Wnt signaling, but the function of Chchd8 is not known. Further experimentation, using over expression and gene silencing demonstrated that LRP6 negatively regulates AHPCs proliferation. Thus, from this study using a system genetics approach, we were able to identify, LRP6 as a novel regulator of adult hippocampal neurogenesis.