The vegetal cortex of the Xenopus oocyte is enriched for several mRNAs critical for early embryonic developmental processes, including germ layer specification and dorsoventral axis formation. A recent microarray screen for other vegetally localized RNAs identified several hundred novel cortex-enriched transcripts, which may have undiscovered roles in early development. In order to better elucidate the functions of localized mRNAs in early development, I characterized the spatiotemporal expression patterns and developmental functions of two novel transcripts, TRIO and F-actin binding protein (triobp) and Cdc42 effector protein 4-like (cep4l). Overexpression and loss-of-function experiments failed to identify a critical role for TrioBP in early Xenopus development. For Cep4l, I found that overexpression of Cep4l induced primary neuron formation throughout the epidermis, preferentially inducing primary sensory neurons. This increase came at the expense of neighboring non-neuronal ciliated and ion-secreting cells, suggesting a role for Cep4l in neural boundary formation. Additionally, I have shown that Cep4l binds specifically to Cdc42 through its known Cdc42/Rac-interactive binding (CRIB) domain, and that this activation was necessary for Cep4l function. Morpholino (MO) oligonucleotide based inhibition of Cep4l protein synthesis resulted in decreased primary sensory neurogenesis. Additionally, I have shown that Cdc42 itself is required for sensory neurogenesis. Furthermore, I find that Fgf8a, an isoform of Fgf8 previously known to regulate neuronal development, but not the Fgf8b isoform, regulates the association of Cep4l and Cdc42. Importantly, I further show that Cep4l and Cdc42 are required for the ability of Fgf8a to induce sensory neurons. Overall, this work suggests a novel role for Cep4l and Cdc42 in the regulation of primary sensory neuronal fate downstream of a unique Fgf8 signaling pathway. I propose that binding of Fgf8a to its receptors activates Cdc42 and recruits Cep4l, which could serve as a scaffold for integrating additional signaling pathways involved in controlling sensory neuron fate.

New neurons are generated throughout adulthood in the dentate gyrus of the hippocampus. The aim of the current study was to address whether differences in the morphological complexity of adult-born granule cells affect their integration into existing dentate gyrus circuitry. To selectively label proliferating cells, we injected a CAG-retrovirus into the dentate gyrus of mice. Either 10, 20, 40, or 80 days following viral infection, mice were injected with pentylenetetrazol (PTZ) to induce hippocampal activation, and expression of the immediate early gene c-fos was used as a marker of activated neurons. We then compared morphological features of neurons across age groups and between Fos+ and Fos- neurons within each age group. We found that dendritic length and branch number increased from 10 to 20 days post infection. Unexpectedly, we also found that dendritic length and branch number decreased from 20 to 40 days post infection, suggesting that the maturation of adult-generated neurons is associated with an active pruning process. Furthermore, we found no significant difference in morphological complexity between Fos+ and Fos- neurons, suggesting that dendritic morphology does not influence integration into dentate gyrus circuitry.

New neurons are generated throughout adulthood in the dentate gyrus of the hippocampus. The aim of the current study was to address whether differences in the morphological complexity of adult-born granule cells affect their integration into existing dentate gyrus circuitry. To selectively label proliferating cells, we injected a CAG-retrovirus into the dentate gyrus of mice. Either 10, 20, 40, or 80 days following viral infection, mice were injected with pentylenetetrazol (PTZ) to induce hippocampal activation, and expression of the immediate early gene c-fos was used as a marker of activated neurons. We then compared morphological features of neurons across age groups and between Fos+ and Fos- neurons within each age group. We found that dendritic length and branch number increased from 10 to 20 days post infection. Unexpectedly, we also found that dendritic length and branch number decreased from 20 to 40 days post infection, suggesting that the maturation of adult-generated neurons is associated with an active pruning process. Furthermore, we found no significant difference in morphological complexity between Fos+ and Fos- neurons, suggesting that dendritic morphology does not influence integration into dentate gyrus circuitry.

In the mature dentate gyrus (DG) new neurons are continuously produced in a process known as adult neurogenesis. These neurons are thought to contribute to learning and memory processes, but their precise function is still not fully understood. The rate of neuronal production and the development of young neurons are affected by stimuli such as physical exercise and learning events. The neurotransmitter γ-aminobutyric acid (GABA) has recently emerged as a possible key mediator of this activity-dependent regulation. My study examined the role of a subtype of GABAA receptors that contains the δ subunit (δGABA(A)R) in the regulation of adult hippocampal neurogenesis. Mice lacking the δ subunit (Gabrd−/− or δ-null mice) displayed decreased maturation, migration and dendritic complexity of adult-born neurons. Conversely, following treatment with a selective δGABA(A)R agonist, neuronal maturation was promoted in wild-type, but not δ-null mice. These results indicate a key role for δGABA(A)R in activity-dependent regulation of adult neurogenesis. When administered to rats, δGABA(A)R agonists promoted neuronal survival as well as maturation. The effect on maturation was blocked by the N-Methyl-D-aspartate (NMDA) blocker AP-5, suggesting that some aspects of δGABA(A)R-mediated regulation require the activation of NMDA receptors. To further understand the contribution of adult born neurons to memory function, neurogenesis in rats was alternatively suppressed using ionizing radiation, or enhanced by allowing the rats to engage in running. The rats were then trained and tested in a behavioral paradigm designed to assess their susceptibility to memory interference, a phenomenon which occurs when similar memories are not sufficiently distinguished from one another. Irradiated rats exhibited increased susceptibility to memory interference, indicating an important role for adult-born neurons in the encoding and/or retrieval of distinct memories. Remarkably, irradiated rats that engaged in running exhibited increased neuronal growth and a complete reversal of the memory impairment,emphasizing the beneficial effects of physical exercise on cognitive functions. Taken together, the findings reported in this dissertation offer novel information about the process of adult neurogenesis and its physiological significance.

In the mature dentate gyrus (DG) new neurons are continuously produced in a process known as adult neurogenesis. These neurons are thought to contribute to learning and memory processes, but their precise function is still not fully understood. The rate of neuronal production and the development of young neurons are affected by stimuli such as physical exercise and learning events. The neurotransmitter γ-aminobutyric acid (GABA) has recently emerged as a possible key mediator of this activity-dependent regulation. My study examined the role of a subtype of GABAA receptors that contains the δ subunit (δGABA(A)R) in the regulation of adult hippocampal neurogenesis. Mice lacking the δ subunit (Gabrd−/− or δ-null mice) displayed decreased maturation, migration and dendritic complexity of adult-born neurons. Conversely, following treatment with a selective δGABA(A)R agonist, neuronal maturation was promoted in wild-type, but not δ-null mice. These results indicate a key role for δGABA(A)R in activity-dependent regulation of adult neurogenesis. When administered to rats, δGABA(A)R agonists promoted neuronal survival as well as maturation. The effect on maturation was blocked by the N-Methyl-D-aspartate (NMDA) blocker AP-5, suggesting that some aspects of δGABA(A)R-mediated regulation require the activation of NMDA receptors. To further understand the contribution of adult born neurons to memory function, neurogenesis in rats was alternatively suppressed using ionizing radiation, or enhanced by allowing the rats to engage in running. The rats were then trained and tested in a behavioral paradigm designed to assess their susceptibility to memory interference, a phenomenon which occurs when similar memories are not sufficiently distinguished from one another. Irradiated rats exhibited increased susceptibility to memory interference, indicating an important role for adult-born neurons in the encoding and/or retrieval of distinct memories. Remarkably, irradiated rats that engaged in running exhibited increased neuronal growth and a complete reversal of the memory impairment,emphasizing the beneficial effects of physical exercise on cognitive functions. Taken together, the findings reported in this dissertation offer novel information about the process of adult neurogenesis and its physiological significance.

Nestin is a type VI intermediate filament protein that marks proliferative cells in the central and peripheral nervous system of vertebrates during development and adulthood. Nestin is not only expressed in progenitor cells of neuronal tissues but is also present in muscle, heart, lung, pancreas and skin follicle tissues. The goal of this thesis is to investigate and characterize the nestin protein in goldfish and relate nestin expression to neuroregeneration and brain plasticity events in the adult goldfish forebrain. Currently little is known about nestin function and regulation in vertebrates, especially in fish. In this study we used Rapid amplification of cDNA ends PCR (RACE-PCR) to isolate goldfish nestin mRNA. We uncovered several different mRNA transcripts. PCR analysis and sequencing further identified three different nestin transcripts of 4003, 2446, and 2126 nucleotides with a predicted protein length of 860, 274, and 344 amino acids respectively. We next applied a multiple-antigenic peptide (MAP) strategy to generate a polyclonal goldfish-specific nestin antibody against a 23 amino acid sequence located at the N-terminal end of goldfish nestin. Western blotting revealed the existence of three different nestin protein isoforms (nestin A, B and C); the first report of nestin isoforms in teleost species. Nestin expression and distribution in the goldfish brain is complex and revealed both individual and tissue-dependent variations. The most remarkable finding following principal component analysis of the western blot data was the uniqueness of the pituitary, hypothalamus and telencephalon. These tissues are proliferative in nature containing progenitor and proliferative cellular pools that are involved in important biological axes such as the motor and reproductive axis. Interestingly, all three tissues were able to change their proliferative cellular profile of nestin protein expression to alleviate the detrimental effects of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) upon administration. The toxin MPTP destroys dopamine neurons in the fish brain leading to motor deficits and reproductive difficulties. The incorporation of 5-bromo-2’-deoxyuriding (BrdU) into newly synthesized DNA revealed an upregulation of BrdU immunolabeling following MPTP administration in the area telencephali pars dorsalis (Vd) and along the ventricular surface area of the telencephalon suggesting the generation of new neurons in the adult central nervous system. This thesis reports novel nestin isoforms and illustrates regenerative events occurring in the goldfish telencephalon following a neurotoxic insult. This work provides a framework for future investigations of the differential roles and regulation of the nestins to better understand seasonal neuronal plasticity, neuronal regeneration and neuronal circuitry in teleost.

Following a stroke there is a significant increase in the number and migration of progenitor cells (PCs) to the infarct, and positive correlations between neurogenesis and recovery. Loss-of-function studies have conflicting findings on whether the ablation of PCs impedes motor or cognitive function post-stroke. This thesis examines if neurogenesis per se is required for motor recovery and spatial learning and memory. PCs were ablated in an adult GFAP-TK rat model that allows for the inducible deletion of GFAP-expressing PCs in the brain. An endothelin-1 (ET-1) stroke was produced and assessment of motor function and spatial learning and memory revealed no differences between control and GFAP-TK rats in which PCs were ablated. This study is the first to use a focal cortical stroke model in a rat to study PCs and stroke recovery, and suggest that PCs and their progeny are dispensable for motor recovery and spatial learning and memory post-stroke.

Ischemic stroke related brain damage causes loss of multiple cell types, including neural and vascular cells. The extent of post-stroke neurogenesis and angiogenesis predicts the level of functional regeneration/recovery after stroke. In this regard, my thesis was focused on defining the molecular process that modulates post-stroke functional recovery by co-ordinating post-stroke neurovascular remodeling. Since stroke-related brain damage releases enriched local microenvironmental cues, I examined the role of a signaling-induced epigenetic pathway, an atypical protein kinase C (aPKC)-mediated phosphorylation of CREB Binding Protein (CBP), in regulating post-stroke neurovascular remodeling and functional recovery. This pathway has previously been shown to be activated by metformin, an adenosine monophosphate kinase (AMPK) activator, to promote the differentiation of neural precursors in the developing and adult brain. Here, I first developed a murine focal cortical ischemic stroke model with persistent motor function deficits by combined intra-cortical injections of endothelin-1 (ET-1) and L-NAME into the sensorimotor cortex. Second, I applied the ET-1/L-Name-induced focal cortical stroke model in a knock-in mouse CBPS436A where the aPKC-CBP pathway is deficient, and showed that the aPKC-CBP pathway is involved in post-stroke functional recovery by coordinating neurovascular remodeling. Specifically, CBPS436A-KI mice displayed reduced motor recovery, correlated with reduced vascular remodeling and impaired post-stroke angiogenesis. Intriguingly, I also observed that CBPS436A-KI mice showed a reduction in the population of stroke-induced newborn pericytes but an increase in the population of perivascularly-derived neural precursors, implying that the aPKC-CBP pathway may be involved in the process that reprograms pericytes into neural precursors. Together, this study elucidates the novel role of the aPKC-CBP pathway in modulating neurovascular remodeling and functional recovery following focal ischemic cortical stroke.

No description available.

The hippocampus has been the focus of memory research for decades. While the functional role of this structure is not fully understood, it is widely recognized as being vital for rapid yet accurate encoding and retrieval of associative memories. Since the discovery of adult hippocampal neurogenesis (AHN) in the dentate gyrus (DG) by Altman and Das in the 1960s, many theories and models have been formulated to explain the functional role it plays in learning and memory. These models postulate different ways in which new neurons are introduced into the DG and their functional importance for learning and memory. Few, if any, previous models have incorporated the unique properties of young adult-born dentate granule cells (DGCs) and their developmental trajectory. In this thesis, we propose a novel computational model of the DG that incorporates the developmental trajectory of these DGCs, including changes in synaptic plasticity, connectivity, excitability and lateral inhibition, using a modified version of the restricted boltzmann machine (RBM). Our results show superior performance on memory reconstruction tasks for both recent and distally learned items, when the unique characteristics of young DGCs are taken into account. The unique properties of the young neurons contribute to reducing retroactive and proactive interference, at both short and long time scales, despite the reduction in pattern separation due to their hyperexcitability. Our replacement model is subsequently extended to support learning dependent regulation of neurogenesis and apoptosis, using a convergence based approach to network growing and pruning. This hybrid additive and replacement model provides a more realistic and flexible approach to investigating the role of neurogenesis regulation in learning and memory. Finally, we incorporate the dentate gyrus model into a full hippocampal circuit to assess cued recall performance. Once again, our neurogenesis model shows decreased proactive and retroactive interference. / Thesis / Master of Science (MSc)