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The hallmark of an adult mammalian central nervous system is the inability to regenerate after an injury. Zebrafish, on the other hand, have an astounding regenerative capacity. After a spinal cord lesion, zebrafish can re-establish the damaged neuronal network and regain their swimming ability within weeks. This is partly due to the presence of the ependymal radial glia (ERGs), which line the wall of the central canal and act as the stem/progenitor cells of the spinal cord. Under homeostatic conditions the ERGs are largely quiescent, however, the lesion triggers them to proliferate and replace cells that have been lost due to the damage. Previous studies have shown that the regeneration of the motor neurons is affected by the signalling pathways similar to those governing the first development of these cells during embryogenesis, such as Sonic hedgehog, Notch and dopamine signalling. Serotonin (5-HT), similar to dopamine, is a monoaminergic neurotransmitter with a wide range of physiological and behavioural functions. It has also been shown to play a role during development of the nervous system. In this doctoral thesis I address the hypothesis that 5-HT has a positive effect on the development and adult regeneration of motor neurons. In addition, I expand on the previously discovered augmenting effect of dopamine on motor neuron development, by analysing the downstream pathways of its action. I show that during the development, incubating embryos in 5-HT increases the proliferation of the motor neuron progenitor (pMN) cells, which leads to augmented motor neuron production. RT-PCR on FAC sorted pMN cells highlights a number of serotonergic receptors that might be responsible for this effect. Although the downstream pathways are still unknown, 5-HT appears not to act on the sonic hedgehog canonical pathway, as shown by the unchanged expression of the hedgehog effector gene, patched2 after 5-HT treatment. I show that 5-HT does not affect the generation of vsx1+ or pax2a+ interneurons, suggesting that it has a predominant effect on motor neuron production. In the intact spinal cord of an adult zebrafish, the pMN-like ERGs express serotonergic receptors, indicating they are responsive to 5-HT stimulation. After a lesion, 5-HT administration enhances the proliferation of the pMN-like ERGs caudal to the lesion site resulting in an increase in the number of newborn motor neurons. Rostral to the lesion site, administration of exogenous 5-HT does not have an effect on the ERG proliferation, possibly due to the fact that the endogenous source of 5-HT, in the form of the descending axons, is still present and might already elicit a maximal response of the progenitor cells. 5-HT does not have an effect on the proliferation of the progenitor cells dorsal or ventral to the pMNlike domain, nor does it affect the regeneration of the serotonergic interneurons. These results suggest that 5-HT from the brain preferentially contributes to the regeneration of the motor neurons. Dopamine is another monoamine shown to enhance motor neuron production during the development and regeneration. To investigate the downstream pathways of dopamine signalling on motor neuron production during embryogenesis, RNA-sequencing was performed on FAC sorted pMN cells after a treatment with a dopamine agonist, pergolide. The results yielded 14 differentially expressed genes (FDR < 0.05) with diverse functions in the cell, indicating that dopamine might act on multiple targets to promote motor neuron production. Taken together, these results demonstrate the positive effect of monoaminergic stimulation on motor neuron development and regeneration. They provide an insight into the pathways that govern the proliferation of stem/progenitor cells in the embryonic and adult spinal cord, which might contribute to the research working on enhancing adult neurogenesis in mammals.

The formation of neurospheres was important in demonstrating that neurogenesis in the adult brain may be fuelled by a stem cell population. The olfactory mucosa is another site of neurogenesis which, in humans, has been observed to contain a stem cell population through the formation of neurospheres (Murrell et al., 2005). Stem cells can be defined as cells capable of self-renewal and multipotency. The aim of this study was to investigate the potential of rat olfactory stem cells growing as neurospheres. The hypothesis is that the rat olfactory mucosa contains a 'true' stem cell population that can be cultured as neurospheres and that will demonstrate multipotency by differentiating into 'non-olfactory' cell types and possess the capacity for self-renewal, if provided with the appropriate environmental niche. Here it was found that adult rat olfactory mucosa is capable of generating neurospheres when cultured in EGF and bFGF. Evidence of self-renewal was provided by the formation of six generations of neurospheres, the formation of neurospheres from single cells and the expression of markers associated with self-renewal by neurosphere cells. The multipotency of olfactory neurosphere cells was demonstrated through manipulation of the stem cell niche. In defined culture conditions, extracellular matrix molecules and growth factors were able to induce the differentiation of neurosphere cells down the dopaminergic lineage pathway. When co-cultured with differentiating cells, neonatal myoblasts and 3T3-L1 cells, olfactory neurosphere cells were able to differentiate and incorporate into a skeletal muscle myotube and differentiate into adipocytes, respectively. In conclusion it was found that the adult rat olfactory mucosa is capable of generating neurospheres. When presented with an appropriate niche neurosphere cells are able to self-renew and demonstrate multipotency.

<p>Many studies on neurogenesis in adult dentate gyrus (DG) have been performed on rodents and other mammalian species, but only a few on adult human DG.  This study is focusing on neurogenesis in adult human DG. To characterize the birth of cells in DG, the expression of the cell proliferation marker Ki67 was examined using immunohistochemistry. Ki67-positive labelling was indeed observed in the granular cell layer and the molecular layer of dentate gyrus and in the hilus of hippocampus, as well as in the subgranular zone (SGZ). The Ki67 positive nuclei could be divided into three groups, based on their morphology and position, suggesting that one of the groups represents neuronal precursors. Fewer Ki67 positive cells were seen in aged subjects and in subjects with an alcohol abuse. When comparing the Ki67 positive cells and the amount of blood vessels as determined by anti factor VIII, no systematic pattern could be discerned. To identify possible stem/progenitor cells in DG a co-labelling with nestin and glial fibrillary acid protein was carried out. Co-labelling was found in the SGZ, but most of the filaments were positive for just one of the two antibodies. Antibodies to detect immature/mature neurons were also used to investigate adult human neurogenesis in DG. The immature marker βIII-tubulin showed a weak expression. The other two immature markers (PSA-NCAM and DCX) used did not work, probably since they were not cross-reacting against human tissue. In summary, this study shows that new cells are continuously formed in the adult human hippocampus, but at a slower pace compared to the rat, and that some of these new cells may represent neuronal precursors.</p>

Many studies on neurogenesis in adult dentate gyrus (DG) have been performed on rodents and other mammalian species, but only a few on adult human DG.  This study is focusing on neurogenesis in adult human DG. To characterize the birth of cells in DG, the expression of the cell proliferation marker Ki67 was examined using immunohistochemistry. Ki67-positive labelling was indeed observed in the granular cell layer and the molecular layer of dentate gyrus and in the hilus of hippocampus, as well as in the subgranular zone (SGZ). The Ki67 positive nuclei could be divided into three groups, based on their morphology and position, suggesting that one of the groups represents neuronal precursors. Fewer Ki67 positive cells were seen in aged subjects and in subjects with an alcohol abuse. When comparing the Ki67 positive cells and the amount of blood vessels as determined by anti factor VIII, no systematic pattern could be discerned. To identify possible stem/progenitor cells in DG a co-labelling with nestin and glial fibrillary acid protein was carried out. Co-labelling was found in the SGZ, but most of the filaments were positive for just one of the two antibodies. Antibodies to detect immature/mature neurons were also used to investigate adult human neurogenesis in DG. The immature marker βIII-tubulin showed a weak expression. The other two immature markers (PSA-NCAM and DCX) used did not work, probably since they were not cross-reacting against human tissue. In summary, this study shows that new cells are continuously formed in the adult human hippocampus, but at a slower pace compared to the rat, and that some of these new cells may represent neuronal precursors.

Autism, a neurodevelopmental disorder, is assumed to result from early neural tube damage. Individuals with Autism exhibit macroencephaly during childhood. To examine increased neurogenesis as a factor in macroencephaly, the valproic acid (VPA) model of Autism was used to examine how exposure to enrichment affects neurogenesis in the dentate gyrus. To induce the model, pregnant rats received two 100mg/kg VPA injections on days 11, 12, and 13 of gestation. Half the pups in each group were exposed to enrichment from post-natal days 30-60. Neurogenesis was examined by fluorescence microscopy for the proliferation marker bromodeoxyuridine (BrdU) and neuronal specific nuclear marker (NeuN). Counts of double-labeled cells were done from the dentate gyrus, an area known for adult neurogenesis. Results indicate that neurogenesis is not abnormal in the VPA model and enrichment increases the neurogenesis similarly in both VPA and control animals. This research provides a better understanding of brain plasticity in the VPA model of Autism.

This thesis reports on a novel use of the Medipix All Resolution System (MARS) Spectral CT scanner as a platform of small animal irradiation. Irradiation from the x-ray source in the scanner was used to inhibit the growth and recovery of neurons in rats. The goal is to extrapolate the relationship between stem cells and memory and functional behaviours. Specific developments were carried out prior to the irradiation of live rats. A live rat holder was designed and built to provide a setup stable enough for fixing the position of the rats head. This was achieved by integrating a three piece bar system into the holder to hold the head from both ears and nose. To quantify the absorbed dose, x-ray exposures were measured using a calibrated ion chamber and were used to generate a depth dose curve with sheets of Perspex layers and radiochromic films. This curve is presumed to simulate the dose inside the rat‘s head. To target a sub region of interest within the rat‘s brain, specific anatomical landmarks were investigated for the design of the lead collimators. The position of the sub volume, the hippocampus, was located through a combination of anatomical landmarks and x-ray transmission images of the rat‘s head. Bregma and the interaural line were used to numerically plot out the co-ordinates of the dorsal and ventral hippocampus which was then translated onto the lead collimators. The x-ray transmission images of euthanized rats were used as a guide to locate the dorsal and ventral hippocampuses. Bregma and the interaural line were the main anatomical landmarks which were used for the design of the lead collimators to be placed around the head. Three pilot rats were irradiated with the designed holder and collimators. The point dose to the hippocampus was calculated using the simulated depth dose curve. Post irradiation status of the neurogenesis was assessed three weeks after the treatment. However only one of the three rats showed a significant reduction in the number of neurons in the hippocampus emphasizing the room for more improvement in the physical setup of the irradiation.

Autism, a neurodevelopmental disorder, is assumed to result from early neural tube damage. Individuals with Autism exhibit macroencephaly during childhood. To examine increased neurogenesis as a factor in macroencephaly, the valproic acid (VPA) model of Autism was used to examine how exposure to enrichment affects neurogenesis in the dentate gyrus. To induce the model, pregnant rats received two 100mg/kg VPA injections on days 11, 12, and 13 of gestation. Half the pups in each group were exposed to enrichment from post-natal days 30-60. Neurogenesis was examined by fluorescence microscopy for the proliferation marker bromodeoxyuridine (BrdU) and neuronal specific nuclear marker (NeuN). Counts of double-labeled cells were done from the dentate gyrus, an area known for adult neurogenesis. Results indicate that neurogenesis is not abnormal in the VPA model and enrichment increases the neurogenesis similarly in both VPA and control animals. This research provides a better understanding of brain plasticity in the VPA model of Autism.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p.149-164