The present study examined social status and adult neurogenesis in the naked mole rat. These animals live in large colonies with a strict reproductive dominance hierarchy; one female and 1-3 males breed, while other members are subordinate and reproductively suppressed. We examined whether social status affects doublecortin (DCX; a marker for immature neurons) immunoreactivity in the dentate gyrus, piriform cortex (PCx), and basolateral amygdala (BLA) by comparing breeders to subordinates. We also examined subordinates removed from their colony and paired with opposite- or same-sex conspecifics for 6 months. Breeders had reduced DCX immunoreactivity in all areas, with BLA effects confined to females. Effects of housing condition were region-specific, with higher PCx DCX immunoreactivity observed in opposite- than same-sex paired subordinates regardless of gonadal status. The opposite pattern was observed in the BLA. Future work will clarify whether findings are attributable to status differences in stress, behavioural plasticity, or life stage.

Les vertébrés, du poisson à l'homme, possèdent un potentiel membranaire médié en partie par les ions chlorure (Cl-). L’une des premières formes d’activité neuronale lors du développement est la dépolarisation médiée par les ions chlorures extrudés par les canaux glycinergiques (GlyR) et GABAergiques. Cette dépolarisation est rendu possible grâce à l’expression retardée du co-transporteur d’ions chlorure et de potassium KCC2 lors du développement qui génère un gradient hyperpolarisant postnatalement chez les mammifères. Le rôle de cette dépolarisation précoce paradoxale durant le développement est inconnu. En injectant l’ARNm de KCC2 dans des embryons de poissons zébrés nouvellement fertilisé, nous avons devancé l’expression de ce co-transporteur rendant ainsi la glycine hyperpolarisante dans tous les neurones dès les premières phases du développement. Nous avons aussi ciblé le récepteur glycinergique directement en bloquant son activité et son expression à l’aide d’une drogue spécifique, la strychnine et d’un morpholino antisens (Knockdown). Dans les trois cas (KCC2, strychnine et GlyR KD), les perturbations de l’activité neuronale ont provoqués des erreurs dans la neurogenèse, en particulier une diminution du nombre d’interneurones sans avoir d’effets sur les motoneurones et les neurones sensoriels. De plus, en bloquant les canaux calciques activés à bas voltage dans le développement avec la drogue nifedipine, il y a des erreurs dans la neurogénèse semblables à celles remarquées dans les trois conditions précédentes. Nous concluons que la dépolarisation précoce par la glycine permet l’entrée du calcium et l’activation de la neurogénèse chez les interneurones. / Vertebrates, from fish to man, have a membrane potential mediated in part by chloride ions (Cl-). One of the first neuronal activity during development of the zebrafish spinal cord is cell depolarisation mediated by chloride extrusion via glycinergic receptors (GlyRs) and GABAergic receptors. This depolarisation is due to the absence of chloride-potassium cotransport channel KCC2, whose expression comes later in development, creating a hyperpolarising gradient. The role of this paradoxal depolarisation period during early stages of development is still unknown. By injecting KCC2 mRNA in newly fertilised zebrafish embryos, we expressed this co-transporter channel in neurons causing glycine to hyperpolarize in early phases of development. We also directly targeted the glycine receptor (GlyR) itself by blocking its activation with a chronic treatment of Strychnine, a specific drug, and by knocking down the expression of this receptor with an antisense morpholino injection. In those three conditions (KCC2, Strychnine and GlyR KD), perturbation of neuronal activity provoked major defects in neurogenesis, particulary in development of interneurons, without affecting other types of cells like motoneurons and sensory neurons. In addition, blocking low-voltage activated calcium channels with nifedipine provoked similar phenotypes. We conclude that the early glycine-mediated depolarisation allow calcium entry, thus activating certain aspects of interneurons neurogenesis.

New neurons are continually produced throughout adult life in the dentate gyrus of the hippocampus, in a process termed adult neurogenesis. Although there is a significant effort in the literature to understand the functional significance of hippocampal neurogenesis, conflicting experimental reports have left the role of neurogenesis unclear. Recently, computational modelling studies have hypothesized that neurogenesis may play a role in allowing association between event and context to be formed in memory. By using a novel odour task and a raised plus maze task, our work demonstrates that the reduction of hippocampal neurogenesis using focal irradiation impairs the ability of animal subjects to utilize contextual information to learn interfering information. The result of this work provides experimental evidence of a unique role neurogenesis may play in learning and memory.

New neurons continue to be added to the dentate gyrus (DG) throughout adulthood and enhancing neurogenesis in this region holds therapeutic potential. However, the molecular mechanisms underlying DG neurogenesis remain elusive. Since developmental and adult neurogenesis often share the same signaling pathways, understanding how the DG develops is crucial to understanding adult neurogenesis. This study aims to determine the role of the retinoblastoma (Rb) protein in DG development and to determine if modulation of this pathway holds potential for enhancing neurogenesis in an adult system. A FoxG1 driven Cre is used to delete Rb in the developing forebrain and the resulting effects are analyzed in in vitro and in vivo mouse models. We show that Rb deletion enhances DG neurogenesis by specifically increasing proliferation of immature neurons. Overall this study suggests that Rb pathway modulation could hold potential for enhancing neurogenesis in the adult.

Hindsight (HNT) is a zinc finger transcription factor that is required for morphogenesis of the Drosophila embryo, having roles in germ band retraction (GBR) as well as dorsal closure (DC). HNT expression is also found in sensory organ precursors (SOP) of the developing pupal peripheral nervous system, and muscle progenitor cells, but the role of HNT in neurogenesis and myogenesis during embryogenesis has not been investigated in any depth. Microarray analysis of embryos over-expressing HNT during GBR and DC identified 1290 genes with significant changes in expression. This data set included many potential HNT targets, including genes associated with myogensis, and a disruption of muscle development was observed in embryos over-expressing HNT. It is possible that HNT may function to repress muscle identity genes in muscle founder cells. In addition, HNT over expressing embryos were found to resemble the neurogenic class of mutants. Among the potential target genes, D-Pax2 (shaven, sparkling, CG11049) expression, which is known to be expressed in the developing peripheral nervous system, was confirmed to be up-regulated following HNT over-expression. Interestingly, D-Pax2 and HNT expression were found to co-localize at the onset of their expression at stages 10-12 in embryos, but were not co-localized in later stages of embryogenesis. The up-regulation of D-Pax2 by HNT over-expression was further characterized and was found to be associated with strong ectopic HNT expression. The relevance of HNT to the regulation of D-Pax2 during normal development remains to be determined, but it is possible that endogenous expression of HNT is involved in D-Pax2 repression.

Cognitive aging is a biological process characterized by physical changes in the brain and subsequent alterations in cognitive function. While neurodegenerative diseases result in extensive neuronal death and anatomical abnormalities, normal aging has subtle changes resulting in a range of cognitive abilities. Early studies of cognitive aging focused on changes in the neuronal population, but evidence has demonstrated that forebrain neurons are largely preserved with age. Furthermore, the proliferation of new neurons in the adult brain has generated great speculation regarding the role and contribution of new neurons to cognitive function. Conversely, both imaging and ultrastructural analyses have shown that age-related alterations in white matter and myelin are good predictors of cognitive impairment, suggesting that alterations in connectivity between brain regions may result in cognitive decline. In this dissertation, a rhesus monkey model of normal aging was used to assess the contribution of adult-neurogenesis and oligodendrogenesis to cognitive function. First, cell proliferation and adult neurogenesis were assessed in the subgranular zone of the hippocampal dentate gyrus. Aged animals demonstrated a decline in proliferating cells and neurogenesis but only limited correlations with behavioral impairment. Immature neurons were also identified in temporal lobe cortices, but results indicate these immature cortical neurons are most likely not adult-generated. Moreover, despite an age-related decline in numbers, they persist throughout the lifespan and many differentiate into Calretinin neurons. Further investigation of white matter alterations used immunohistochemistry and diffusion spectrum imaging to correlate oligodendrocyte numbers with white matter connectivity. In the corpus callosum and cingulum bundle, there were no correlations with age, but cognitive impairment was associated with increased oligodendrocyte number and decreased white matter connectivity. These correlations were only present in the anterior aspect of the cingulum bundle, not the posterior cingulum suggesting differential oligodendrocyte responses along the anterior-posterior axis of the brain. Together, these data demonstrate an age-related decline in adult neurogenesis may be only a small contributor to cognitive impairment. Additionally, a reserve pool of immature neurons continues to differentiate in the temporal cortex potentially contributing to local plasticity. Furthermore, cognitive impairment rather than aging has a stronger correlation with oligodendrocytes alterations and connectivity.

The loss of neurons throughout aging, due to trauma or neurodegenerative diseases, was considered irreversible until recent discoveries demonstrated the capacity for the postnatal mammalian brain to generate new neurons in discrete niches capable of integrating into existing neural circuitry. Adult neurogenesis is highly dynamic and modulated by numerous factors. However, the temporal patterns of adult neurogenesis, kinetics of cell proliferation, and migration remain poorly understood. Zebrafish, a model used in this investigation, is a diurnal vertebrate with a circadian clock and clock-controlled processes organized similar to humans. Importantly, zebrafish display active neurogenesis. The studies comprising this dissertation demonstrate for the first time that a diurnal vertebrate displays robust circadian, i.e. near-24-h, patterns of adult neurogenesis. It proceeds as an orderly transition of cells from G1 to S phase of the cell cycle throughout the day, followed by nighttime progression of G2 phase, culminating with M phase in the early morning. While all five neurogenic niches studied reveal a common circadian pattern, each niche demonstrates a distinct S length and timing of the G1-S transition. Further investigation into kinetics of adult neurogenesis focused on the events occurring in the neurogenic niches over several days. Both experimental and mathematical modeling approaches determined a consistent number of neural stem cells (NSCs) dividing daily. These approaches also elucidated the predominant modes of division for transient amplifying cells, the neural progenitors (NPCs), the pace of migration and survival of their progeny. Finally, this dissertation addressed age-related changes in adult neurogenesis in zebrafish, supporting its gradual decline with age. Developing a pathological aging model, based on excessive nutrition throughout development and maturation, revealed a major decline in the number of dividing NSCs and extreme modification of the pattern of division and survival of NPCs. Together the results of the studies presented in this dissertation reveal that adult neurogenesis undergoes predictable dynamic changes over hours, days, and years. Future studies using a high-throughput zebrafish model should provide needed insights into the role of specific factors in adult neurogenesis and help develop therapeutic strategies to benefit human patients.

In the 1980s, it was widely accepted that new neurons are continuously generated in the dentate gyrus of the mammalian hippocampus. Since its acceptance, researchers have employed various techniques and behavioral paradigms to study the proliferation, differentiation, and functional role of adult-born neurons. This literature thesis aims to discuss how optogenetics is able to overcome the limitations of past techniques and provide the field with new insights into the functional role of neurogenesis. We will review the current knowledge on both adult hippocampal neurogenesis and optogenetics, present representative studies using optogenetics to investigate neurogenesis and discuss potential limitations and concerns involved in using optogenetics.

Forebrain development is controlled by a set of transcription factors which are expressed in dynamic spatiotemporal patterns in the embryonic forebrain and are known to regulate complex gene networks. Pax6 is a transcription factor that regulates corticogenesis and mutations affecting Pax6 protein levels cause neurodevelopmental defects in the eyes and forebrain in both humans and mice. In previous studies, it was shown that the graded expression pattern of Pax6 protein, which is high rostro-laterally to low caudo-medially in the cerebral cortex, is critical for its control of cell cycle progression and proliferation of cortical progenitors. However the underlying mechanisms are still unclear. Based on a microarray analysis carried out in our laboratory, a number of cell cycle-related candidate genes that may be affected by Pax6 have been identified. One such gene, Cell division cycle associated 7 (Cdca7) is expressed in a counter-gradient against that of Pax6. In my current study, I found that Cdca7 mRNA expression in the telencephalon is upregulated in Pax6 null (Small eye) mutants and downregulated in mice that overexpress PAX6 (PAX77) across developing time points from E12.5 to E15.5. There are several potential Pax6 binding motifs located in the genomic locus upstream of Cdca7. However, by chromatin immunoprecipitation, it is showed that none of the predicted binding sites are physically bound by Pax6. Promoter luciferase assays using fragments combining five suspected binding motifs show that Pax6 is functionally critical. Cdca7 is also identified as a Myc and E2F1 direct target and is upregulated in some tumours but its biological role is not fully understood. Current work using in utero electroporation to overexpress Cdca7 around the lateral telencephalon, where Cdca7 expression levels are normally low, tested the effects on the proliferation and differentiation of cortical progenitor cells in this region. In E12.5 mice embryos, overexpression of Cdca7 protein causes fewer intermediate progenitor cells and post-mitotic neurons to be produced but these effects were not found in E14.5 embryos. This result implies that Cdca7 may affect cell fate decision during cortical development.

abstract: Though for most of the twentieth century, dogma held that the adult brain was post-mitotic, it is now known that adult neurogenesis is widespread among vertebrates, from fish, amphibians, reptiles and birds to mammals including humans. Seasonal changes in adult neurogenesis are well characterized in the song control system of song birds, and have been found in seasonally breeding mammals as well. In contrast to more derived vertebrates, such as mammals, where adult neurogenesis is restricted primarily to the olfactory bulb and the dentate gyrus of the hippocampus, neurogenesis is widespread along the ventricles of adult amphibians. I hypothesized that seasonal changes in adult amphibian brain cell proliferation and survival are a potential regulator of reproductive neuroendocrine function. Adult, male American bullfrogs (Rana catesbeiana; aka Lithobates catesbeianus), were maintained in captivity for up to a year under season-appropriate photoperiod. Analysis of hormone levels indicated seasonal changes in plasma testosterone concentration consistent with field studies. Using the thymidine analogue 5-bromo-2-deoxyuridine (BrdU) as a marker for newly generated cells, two differentially regulated aspects of brain cell neogenesis were tracked; that is, proliferation and survival. Seasonal differences were found in BrdU labeling in several brain areas, including the olfactory bulb, medial pallium, nucleus accumbens and the infundibular hypothalamus. Clear seasonal differences were also found in the pars distalis region of the pituitary gland, an important component of neuroendocrine pathways. BrdU labeling was also examined in relation to two neuropeptides important for amphibian reproduction: arginine vasotocin and gonadotropin releasing hormone. No cells co-localized with BrdU and either neuropeptide, but new born cells were found in close proximity to neuropeptide-containing neurons. These data suggest that seasonal differences in brain and pituitary gland cell neogenesis are a potential neuroendocrine regulatory mechanism. / Dissertation/Thesis / M.S. Biology 2012