Les enfants qui ont subi une asphyxie périnatale modérée (MPA) risquent de développer des déficits cognitifs et comportementaux subtils et durables, notamment des troubles d'apprentissage et des problèmes émotionnels. Comprendre les mécanismes sous-jacents est une étape essentielle pour concevoir une thérapie ciblée. Déterminer comment le développement du cerveau est corrélé entre les humains et les rongeurs n'est pas simple, mais il existe également un alignement inter-espèces considérable en termes d'étapes clés du développement. Sur la base des changements biochimiques et neuroanatomiques au cours du développement précoce, le consensus général est qu'un cerveau de rongeur P8-10 correspond à peu près au cerveau d'un enfant à terme ; par conséquent, nous avons utilisé cette fenêtre temporelle comme référence pour développer un modèle préclinique de MPA chez la souris. Nous avons d'abord établi un protocole qui nous permet d'observer de manière fiable les crises induites par l'hypoxie chez les souris postnatales. Nous avons constaté que l'exposition de chiots P8-9 directement à 4 % d'O2 pendant 8 minutes induit de manière fiable des crises avec une latence d'environ 5 minutes chez 3 souches de souris (FVB, C57Bl/6, 129S6). Cet aspect est cliniquement pertinent car les convulsions sont la caractéristique néonatale la plus importante de l'encéphalopathie de stade 2 (modérée) telle que définie par l'échelle de Sarnat. Les souris MPA adultes présentent des séquelles à long terme sur des performances cognitives spécifiques, notamment des déficits de la mémoire de reconnaissance et de la flexibilité cognitive, mais aucune altération du comportement moteur et émotionnel. Le cortex préfrontal (PFC) régule la flexibilité cognitive et le comportement émotionnel. Les neurones qui libèrent la sérotonine (5-HT) projettent vers le PFC, et les composés modulant l'activité 5-HT influencent l'émotion et la cognition. On ne sait pas si les dérégulations de la 5-HT contribuent aux problèmes cognitifs induits par le MPA. Dans une première étude, nous avons trouvé que les niveaux d'expression de 5-HT, quantifiés par immunohistochimie, et de libération de 5-HT, quantifiés par microdialyse in vivo chez des souris éveillées, sont réduits dans le PFC de souris MPA adultes. Les souris MPA présentent également une régulation de la température corporelle altérée après l'injection de l'agoniste des récepteurs 5-HT1A, 8-OH-DPAT, suggérant la présence de déficits dans la fonction des auto-récepteurs 5-HT sur les neurones du raphé. Enfin, le traitement chronique de souris MPA adultes avec de la fluoxétine, un inhibiteur du transporteur de recapture de la 5-HT, ou l'agoniste des récepteurs 5-HT1A, la tandospirone, sauve la flexibilité cognitive et les troubles de la mémoire. Ensemble, ces données démontrent que le développement de la fonction du système 5-HT est vulnérable à une asphyxie périnatale modérée. L'hypofonctionnement de la 5-HT pourrait à son tour contribuer à une déficience cognitive à long terme à l'âge adulte, indiquant une cible potentielle pour les thérapies pharmacologiques. Les circuits GABAergiques comprennent une variété étonnante de différents types de cellules, qui sont probablement recrutées par différents événements comportementaux. Un sous-type important de cellules GABAergiques, les cellules positives à la parvalbumine (PV), génèrent des potentiels d'action à haute fréquence et synchronisent l'activité des neurones pyramidaux excitateurs. Les cellules PV sont particulièrement importantes pour la génération d'oscillations gamma, qui à leur tour régulent de nombreuses fonctions cognitives, notamment le traitement attentionnel axé sur les objectifs et la mémoire de travail. Des découvertes récentes indiquent que les cellules PV utilisent beaucoup plus d'énergie que les autres neurones corticaux, ce qui peut les rendre très vulnérables aux conditions de stress métabolique et oxydatif causées par le MPA. Nos données ont montré que l'expression de PV est altérée chez les souris MPA adultes. Nous avons en outre constaté que le niveau d'expression du récepteur de la neurotrophine p75NTR, qui limite la maturation des cellules PV au cours de la première semaine postnatale, est augmenté chez les souris MPA. La suppression génétique de p75NTR dans les neurones GABAergiques exprimant le facteur de transcription Nkx2.1, qui comprend les cellules PV, protège les souris de la perte de niveaux de PV et des effets cognitifs à long terme du MPA. Enfin, un traitement d'une semaine avec un inhibiteur de p75NTR commençant après le MPA sauve complètement les déficits d'activité cognitive et corticale chez les souris adultes. L'ensemble de ces données révèle une cible moléculaire potentielle pour le traitement des altérations cognitives causées par le MPA. / Children who experienced moderate perinatal asphyxia (MPA) are at risk of developing long lasting subtle cognitive and behavioral deficits, including learning disabilities and emotional problems. Understanding the underlying mechanisms is an essential step for designing targeted therapy. Determining how brain development correlates between humans and rodents is not straightforward, however there is also considerable cross-species alignment in terms of key developmental milestones. Based on biochemical and neuroanatomical changes during early development, the general consensus is that a P8-10 rodent brain corresponds roughly to the brain of a term infant; therefore, we used this time window as reference to develop a preclinical model of MPA in mouse. We first established a protocol that allows us to reliably observe hypoxia-induced seizures in postnatal mice. We found that exposing P8-9 pups directly to 4% O2 for 8 minutes reliably induces seizures with a latency of about 5’ in 3 mouse strains (FVB, C57Bl/6, 129S6). This aspect is clinically relevant as seizures are the most prominent neonatal hallmark of Stage 2 (Moderate) encephalopathy as defined by the Sarnat Scale. Adult MPA mice show long-term sequelae on specific cognitive performance, including deficits in recognition memory and cognitive flexibility, but no impairment in motor and emotional behavior. The prefrontal cortex (PFC) regulates cognitive flexibility and emotional behavior. Neurons that release serotonin (5-HT) project to the PFC, and compounds modulating 5-HT activity influence emotion and cognition. Whether 5-HT dysregulations contribute to MPA-induced cognitive problems is unknown. In a first study, we found that 5-HT expression levels, quantified by immunohistochemistry, and 5-HT release, quantified by in vivo microdialysis in awake mice, are reduced in PFC of adult MPA mice. MPA mice also show impaired body temperature regulation following injection of the 5-HT1A receptor agonist 8-OH-DPAT, suggesting the presence of deficits in 5-HT auto-receptor function on raphe neurons. Finally, chronic treatment of adult MPA mice with fluoxetine, an inhibitor of 5-HT reuptake transporter, or the 5-HT1A receptor agonist tandospirone rescues cognitive flexibility and memory impairments. All together, these data demonstrate that the development of 5-HT system function is vulnerable to moderate perinatal asphyxia. 5-HT hypofunction might in turn contribute to long-term cognitive impairment in adulthood, indicating a potential target for pharmacological therapies. GABAergic circuits comprise an astonishing variety of different cell types, which are likely recruited by different behavioral events. An important subtype of GABAergic cells, the fast-spiking, parvalbumin-positive (PV) cells, generate action potentials at high frequency and synchronize the activity of excitatory pyramidal neurons. PV cells are particularly important for the generation of gamma oscillations, which in turn regulate many cognitive functions including goal-directed attentional processing and working memory. Recent findings indicate that PV cells utilize much more energy than other cortical neurons, which may render them highly vulnerable to conditions of metabolic and oxidative stress caused by MPA. Our data showed that PV expression is impaired in adult MPA mice. We further found that the expression level of the neurotrophin receptor p75NTR, which limits PV cell maturation during the first postnatal week, is increased in MPA mice. Genetic deletion of p75NTR in GABAergic neurons expressing the transcription factor Nkx2.1, which include PV cells, protects mice from PV levels loss and the long-term cognitive effects of MPA. Finally, one week treatment with a p75NTR inhibitor starting after MPA completely rescues the cognitive and cortical activity deficits in adult mice. All together this data reveals a potential molecular target for the treatment of the cognitive alterations caused by MPA.

Fung Kwai Ching. / "August 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 188-205). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Previous research has indicated that the perirhinal cortex (PRh) and posterior parietal cortex (PPC) functionally interact to mediate crossmodal object representations in rats; however, it remains to be seen whether other cortical regions contribute to this cognitive function. The prefrontal cortex (PFC) has been widely implicated in crossmodal tasks and might underlie either a unified multimodal or amodal representation or comparison mechanism that allows for integration of object information across sensory modalities. The hippocampus (HPC) is also a strong candidate, with extensive polymodal inputs, and has been implicated in some aspects of object recognition. A series of lesion based experiments assessed the roles of HPC, PFC and PFC sub regions [medial prefrontal (mPFC) and orbitofrontal cortex (OFC)], revealing functional dissociations between these brain regions using two versions of crossmodal object recognition: 1. spontaneous crossmodal matching (CMM), which requires rats to compare between a stored tactile object representation and visually-presented objects to discriminate the novel and familiar stimuli; and 2. crossmodal object association (CMA), in which simultaneous pre-exposure to the tactile and visual elements of an object enhances CMM performance across long retention delays. Notably, while inclusive PFC lesions impaired both CMM and CMA tasks, selective OFC lesions disrupted only CMM, whereas selective mPFC damage did not impair performance on either task. Furthermore, there was no impact of HPC lesions on either CMM or CMA tasks. Thus, the PFC and the OFC play a selective role in crossmodal object recognition but the exact contributions and interactions of the regions will require further research to elucidate. / PDF Document / Natural Sciences and Engineering Research Council of Canada (NSERC)

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Abstract In the Cape Fynbos of South Africa, Psoralea pinnata (L.) plants occur naturally in both wetland and welldrained soils and yet effectively fix N2 under the two contrasting conditions. In this study, nodule structure and functioning in P. pinnata plants from the two habitats were evaluated using light and transmission electron microscopy (TEM), as well as the 15N natural abundance technique. The results showed that, structurally, fully developed P. pinnata nodules were spherical in shape with six components (namely, lenticels, periderm, outer cortex, middle cortex, inner cortex, and a central bacteria-infected medulla region). Morphometric analysis revealed 44 and 84 % increase in cell area and volume of wetland nodules compared to those from upland. The percentage area of nodules occupied by the middle cortex in wetland nodules was twice that of upland nodules. As a result, the size of the medulla region in wetland nodules was significantly reduced compared to upland nodules. Additionally, the average area of medulla occupied by intercellular air spaces in wetland nodules was about five times that of upland nodules (about 431 % increase in wetland over upland nodules). TEM data also showed more bacteroids in symbiosomes of upland nodules when compared to wetland nodules. However, isotopic analysis of above-ground plant parts revealed no differences in symbiotic parameters such as N concentration, ∂15N and %Ndfa between wetland and upland P. pinnata plants. These results suggest that, under limiting O2 conditions especially in wetlands, nodules make structural and functional adjustments to meet the O2 demands of N2-fixing bacteroids.

Bipolar affective disorder and schizophrenia are highly heritable psychiatric illnesses and the leading causes of worldwide disability. The orbitofrontal cortex (OFC) is a region of the frontal lobe with wide spread connectivity with other brain areas involved in reward, motivation and emotion. Evidence from various neuroimaging, genetic, post-mortem and brain lesion studies suggest that orbitofrontal cortex may play a role in pathophysiology of mental illnesses. This thesis sought to investigate the pathogenesis of major psychiatric illnesses through the investigation of orbitofrontal morphology in schizophrenia and bipolar disorder and through its associations with brain structure and function. Orbitofrontal morphology and its structural and functional associations were examined in healthy controls, patients with schizophrenia or bipolar affective disorder, and those at high genetic risk using functional and structural MRI. In the first study we found that the orbitofrontal type III is more frequent and the orbitofrontal type I is less common in the right hemisphere in patients with schizophrenia while in patients with bipolar disorder type III appears more often in both left and right hemispheres. We then sought to examine the relationship of orbitofrontal morphology to disease risk in a study of 146 people at high risk of developing schizophrenia and 110 people at high risk of developing bipolar disorder. We discovered that in the unaffected high risk groups the orbitofrontal type III predicted the development of later psychiatric illnesses, when combined with anterior cingulate morphology. Finally we showed, in a further study, that OFC morphology was associated with measures of schizotypy, brain structure, brain function and cognition. In conclusion, orbitofrontal morphology is linked to major psychiatric disorder and has significant structural and functional associations. As orbitofrontal sulcogyral patterns are formed in early life a fuller awareness of their relevance to brain function holds out the prospect that we could use such measures as an indicator of vulnerability to the development of illness later in life. This work points to the potential for the foundation of a theory of predictive associations between morphological patterns and the development of psychosis.

The adrenal cortex is classically divided into three morphologically and biochemically distinct zones, covered by a thin, cellular capsule. The adult adrenal cortex is a dynamic tissue in which distinct regions of cell proliferation, movement and death have been identified. Several models for stem cell maintenance of the adult adrenal cortex have been proposed, but adrenocortical stem cells have not yet been identified. Adrenal cortices of 21OH/LacZ transgenic mice show similar mosaic patterns of β-galactosidase staining to X- inactivation mosaics and LacZ ↔ wildtype chimeras. 21OH/LacZ mice provide a tool for lineage analysis, which may help to i) identify clones of cells produced by stem cells in the adult, ii) determine when stem cells begin to function and iii) evaluate different models of how stem cells maintain the adrenal cortex. Analysis of 21OH/LacZ transgenic adrenal cortices showed that the randomly orientated clusters of fetal patches change progressively during the perinatal period to adult radial stripes. Correlation of changes in mosaic patterns and the locations of cell proliferation suggests that the stripes arise by edge-biased growth during the perinatal growth period. Although stem cells may not be involved in the initial formation of stripes, it seems likely that stem cells later maintain the stripes by producing clones of cells that move centripetally to displace the earlier fetal patterns and later replace aging cells. Various combinations of BrdU labelling and chase periods demonstrated that most cell division occurred in the outer 40% of the adrenal cortex, confirmed that cells moved towards the medulla and identified a population of label-retaining cells near the capsule, which could include stem cells. (Stem cells have been recognised as BrdU label-retaining cells in other tissues because they divide less frequently than their daughter cells so dilute the incorporated BrdU more slowly.) Stripe patterns in adult 21OH/LacZ transgenic adrenal cortices were examined to try to distinguish between various models proposed for stem cell maintenance of the adrenal cortex. The observed continuous radial stripe pattern favours the general hypothesis that a single population of stem cells in the periphery maintains the entire adrenal cortex, although other explanations are possible. Quantitative analysis of adult stripe patterns did not show the reduction in stripe number that might be predicted if an age-related decline in adrenocortical stem cell function occurs, as may happen in some other tissues.

The adrenal gland is an important endocrine organ, protecting the body against acute and chronic stress. The adrenal cortex consists of three morphologically and functionally distinct zones: the outer zona glomerulosa (zG), the zona fasciculata (zF), and the innermost zona reticularis (zR). In rodents, zG cells produce mineralocorticoids (mainly aldosterone), while zF cells secrete glucocorticoids (mainly corticosterone). The functions of zG and zF are defined by the mutually exclusive expression of Cyp11b2 and Cyp11b1 that encode the enzymes aldosterone synthase and 11β-hydroxylase, which catalyze the terminal reactions in the production of aldosterone and corticosterone, respectively. This thesis aims to investigate the maintenance of the definitive mouse adrenal cortex. This involves studies to identify the location of adrenal stem/progenitor cells, and the mechanisms by which differentiated adrenocortical cells are replenished in the adult mice. BrdU pulse-chase studies provided valuable information about cell division and cell fate under physiological or pathophysiological stimulations. The distribution of adrenocortical cells with nuclei stained positively for BrdU and/or Ki67 was identified. Ki67 labelling marked actively dividing cells and showed that adrenocortical cells originate at or around the zG/zF interface. BrdU labelling indicated that, following cell division, cells are displaced inwards and outwards. Acute angiotensin II treatment was shown to have no significant effects on the cell proliferation or turnover in any of the adrenocortical zones. The pathophysiological effects of long-term ACTH treatment were analyzed in a mouse model of congenital adrenal hyperplasia caused by a null mutation of Cyp11b1. Cell hypertrophy was evident in all regions of the adrenal cortex due to the impaired negative-feedback of the HPA axis. Adrenocortical cell proliferation was also increased particularly in the outer zona fasciculata at the border between zG and zF where adrenocortical stem/progenitor cells might be located. The intervening steps between cell proliferation and the final differentiation into steroidogenic zG and zF cells have yet to be discovered. A visual method of monitoring levels of Cyp11b2 and Cyp11b1would offer a convenient approach to track the stages of adult stem cell differentiation that lead to normal adrenal maintenance in vivo and in vitro. In the present study an AS-mCherry-11B-EGFP BAC construct was successfully engineered, in which Cyp11b2 and Cyp11b1 were substituted by mCherry and EGFP, respectively. This BAC construct was characterized in mouse adrenocortical Y1 cells. It was determined that EGFP faithfully recapitulated the expression of Cyp11b1. Forskolin or cAMP treatment induced a rapid cell rounding effect and caused the increased expression of EGFP transgene and endogenous Cyp11b1. An attempt was made to establish a transgenic mouse model, in which zG and zF cells would be marked with mCherry and EGFP respectively, allowing the differentiation of an adrenocortical stem cell to be traced. Following microinjection of the BAC into mouse zygotes, twoAS-mCherry-11B-EGFP transgenic founder mice were identified. Unfortunately, neither of them was able to transmit the transgene through germline, suggesting the mosaicism of transgene integration. Indeed, mosaicism of the transgenic adrenals was demonstrated by RT-PCR and immunostaining, which also revealed that the exogenous EGFP expression faithfully recapitulated the endogenous Cyp11b1 in adrenals. Although it is assumed that expression of Cyp11b2 and Cyp11b1 are mutually exclusive, zG and zF cells may have the plasticity to allow the transition from one cell type into another. The AS-mCherry-11B-EGFP BAC construct is a useful tool for studying in vitro ES cell differentiation towards the adrenocortical lineage. Transgenic AS-mCherry-11B-EGFP ES cells were successfully differentiated into mesenchymal stem cells, as identified by the expression of molecular markers for the mesenchymal lineage. It has been reported that steroidogenic factor (Sf1) can promote the differentiation of MSCs into steroidogenic cells, and Shh plays an important role in Sf1 expression and the consequent adrenal development. However, Shh treatment failed to achieve transformation of mesenchymal cells into adrenocortical cells. It is thought there might be a requirement for additional factors to combine with Shh in promoting the transdifferentiation of ESC-derived mesenchymal cells. Future studies will focus on the genetic control of Cyp11b1 and Cyp11b2 in transgenic AS-mCherry-11B-EGFP ES cells. In conclusion, the location and fate of the adrenocortical progenitor cells were demonstrated by the BrdU pulse-chase studies in different mouse models. An AS-mCherry-11B-EGFP BAC construct was generated, and used to study the mutual and differential controls of Cyp11b1 and Cyp11b2 expression in adrenocortical cells in vitro and in transgenic mice in vivo.

This thesis provides insights into the mechanism by which thalamocortical axons (TCAs) approach the cortex from their origin in the thalamus. Previous studies suggested that the reciprocal projections from the prethalamus and the ventral telencephalon guide TCAs to descend through the prethalamus and cross the diencephalic-telencephalic boundary (DTB), after which TCAs navigate through permissive corridor cells in the ventral telencephalon and cross the pallial-subpallial boundary (PSPB) before reaching their final targets in the cortex. The ‘Handshake Hypothesis’ proposed that pioneer axons from cortical preplate neurons guide TCAs into corresponding cortical areas. However, there is a lack of convincing evidence on whether TCAs need any guidance to cross the PSPB. In the current study, Adenomatous polyposis (Apc) gene is conditionally deleted from the cortex, by using Emx1Cre-APCloxP recombination technology. Apc is widely expressed in the nervous system including the cortical plate of the cortex and regulates axonal growth and neuronal differentiation. Deleting Apc may block neurite extension and/or affect the formation of attractive or repulsive cues in the cortex. By using DiI tracing as well as L1 immunohistochemistry techniques, I showed that in the Apc mutants cortical axons are absent and that TCAs initially navigate into the ventral telencephalon normally but fail to complete their journey into the cortex. They stop as they approach the PSPB, although the PSPB doesn’t seem to be directly affected by the mutation of Apc in the cortex. Additionally, Ig-Nrg1 (Neuregulin-1), the secreted protein that was suggested to play long-range roles in attracting TCAs towards the cortex, is present in the Apc mutant. This implies that Ig-Nrg1 is not sufficient for guiding TCAs into the cortex, and that additional guidance factors are needed. Moreover, my in vitro explant culture experiments show that the mutant cortex neither repel nor inhibit thalamic axonal outgrowth, indicating that the failure of TCAs in reaching the cortex is not due to the change of repulsive cues secreted by the mutant cortex. It rather indicates that the guidance factors for TCAs are likely to function through cell-cell contact mediated mechanisms. The Apc mutant cortex lacks these guidance factors, which might be the cortical axons. In conclusion, my data reveal a choice point for TCAs at the PSPB. Guidance factors from the cortex are needed for TCAs to cross the PSPB, which are absent in the Apc mutant. TCAs may need the direct contact with cortical axons and use them as an axonal scaffold to navigate into the cerebral cortex.

Hippocampus has an extended developmental trajectory, with refinements occurring in the trisynaptic circuit until adolescence. While structural change should suggest a protracted course in behavior, some studies find evidence of precocious hippocampal development in the first postnatal year and continuity in memory processes beyond. However, a number of memory functions, including binding and relational inference, can be cortically supported. Evidence from the animal literature suggests that tasks often associated with hippocampus (visual paired comparison, binding of a visuomotor response) can be mediated by structures external to hippocampus. Thus, a complete examination of memory development will have to rule out cortex as a source of early memory competency. We propose that early memory must show properties associated with full function of the trisynaptic circuit to reflect "adult-like" memory function, mainly (1) rapid encoding of contextual details of overlapping patterns, and (2) retention of these details over sleep-dependent delays. A wealth of evidence suggests that these functions are not apparent until 18-24 months, with behavioral discontinuities reflecting shifts in the neural structures subserving memory beginning approximately at this point in development. We discuss the implications of these observations for theories of memory and for identifying and measuring memory function in populations with typical and atypical hippocampal function. (C) 2015 The Authors. Published by Elsevier Ltd.