Myelin is essential for normal nervous system conduction as well as providing metabolic support for the ensheathed axon and has been implicated to influence axon calibre (diameter of the axon body) growth. In demyelinating diseases, the disruption of these functions causes axon degeneration resulting in neurological impairment. The neurons that are myelinated in the CNS and the axon-oligodendrocyte (axon- OL) interactions that might regulate axon calibre and myelination during myelinated axon formation are still mostly unknown, preventing a deeper understanding of CNS development and repair. This doctoral thesis identifies a specific subset of interneurons that are myelinated and investigates the axon-oligodendrocyte interactions during axon calibre growth and initial myelination. In the zebrafish spinal cord, Commisural Primary Ascending interneurons (CoPA), Circumferential Descending interneurons (CiD) and reticulospinal neurons are amongst the first to be myelinated, whereas Commisural Bifurcating Longitudinal interneurons (CoBL) and Circumferential Ascending interneuron (CiA) are not myelinated during early developmental stages. Of the myelinated neurons, axon calibre of reticulo spinal neurons is increased in time with myelin ensheathment, while the axon calibre of CoPA and CiD interneurons is not increased with the onset of myelination. In order to investigate whether there might be a causative relationship between axon calibre increase and myelin ensheathment, the majority of oligodendrocytes were eliminated by olig2 morpholino knockdown. In the absence of oligodendrocytes, the axon calibre of reticulospinal neurons was normal, demonstrating that axon calibre growth is independent of axon-OL interactions and myelin ensheathment. In order to further investigate which aspects of myelinated axon formation might be regulated by axon-OL interactions, axonal activity was reduced through inhibition of synaptic vesicle release by global expression of Tetanus-toxin (TetTx). TetTx treated zebrafish showed a 40% decrease of myelinated axons in the spinal cord. Interestingly, only 10% of this reduction was caused by a decrease in oligodendrocyte number in the spinal cord. Single cell analysis of individual oligodendrocytes revealed a 30% reduction of myelin sheaths per oligodendrocyte in TetTx treated animals, indicating a positive correlation between synaptic vesicle release and the extent of myelination. Timelapse analysis of the myelinating behaviour of individual oligodendrocytes revealed that the decrease in myelin sheaths per cell in the absence of synaptic vesicle release results from a reduction in the initial formation of sheaths rather than an increased retraction of myelin sheaths. Furthermore, individual myelin sheaths formed by the same oligodendrocyte exhibit a dynamic range of different growth rates in control animals, which was reduced to a more uniform, slow growth of myelin sheaths in the absence of synaptic vesicle release. This suggests that local axon-OL interactions can regulate the dynamic myelin sheath growth through synaptic vesicle release. The analyses in this doctoral thesis identifies a subset of the neurons that are myelinated during the onset of myelination in the zebrafish spinal cord, demonstrates that axon caliber growth of these neurons is independent of myelin ensheathment and that axon-OL interactions mediated by synaptic vesicle release can regulate the extent of myelination and influence the dynamic myelinating behavior of oligodendrocytes in vivo. These findings begin to elucidate the axon-OL interactions underlying myelinated axon formation during CNS development, from which future studies might derive neuro-regenerative treatments for demyelinating diseases.

No description available.

In addition to acting in synaptic transmission, neurotransmitters have been shown to play roles in the development of nervous system. Developing neurons extend neurites to connect to their target cells, and growth cones at the tip of growing neurites are critical for pathfinding. Although evidence for the regulation of axonal growth and growth cone guidance by neurotransmitters and neuromodulators is emerging, less is known about the mechanisms by which neurotransmitters affect developing neurons. Here, I focus on three neurotransmitters/ neuromodulators and describe their actions (a) at the level of growth cone, especially on filopodia, which serve as sensors that allow growth cones to probe the environment they are traversing, and (b) on how neurotransmitters modulate neuronal electrical properties, which, in itself, have been shown to affect neurite extension. The goals of this dissertation are to investigate 1) the cholinergic modulation of neuronal activity and its effects on growth cone motility; 2) the excitatory modulation of neuronal excitability by nitric oxide (NO); and 3) the inhibitory modulation of neuronal activity by dopamine (DA). The work uses a well-established model system to investigate growth cone motility and neuronal activity: identified neurons from the pond snail Helisoma trivolvis studied in cell culture or in the intact ganglion in situ. The study of B5 neurons demonstrates that acetylcholine (ACh) induces filopodial elongation, which is mediated by opening of nicotinic ACh receptors, membrane depolarization, and elevation of intracellular Ca level in growth cones. This dissertation also shows that NO inhibits two types of Ca-activated K channels to depolarize the membrane potential of B19 neurons. Additionally, the study reveals that DA serves as an inhibitory neurotransmitter to hyperpolarize and silence the electrical activity of firing B5 neurons via a D2-like receptor/PLC/K channel pathway. Taken together, this dissertation elucidates novel cellular mechanisms through which neurotransmitters can regulate growth cone motility and neuronal electrical properties, further supporting evidence for potential roles of neurotransmitters in axon pathfinding and synaptic transmission in vivo.

In addition to acting in synaptic transmission, neurotransmitters have been shown to play roles in the development of nervous system. Developing neurons extend neurites to connect to their target cells, and growth cones at the tip of growing neurites are critical for pathfinding. Although evidence for the regulation of axonal growth and growth cone guidance by neurotransmitters and neuromodulators is emerging, less is known about the mechanisms by which neurotransmitters affect developing neurons. Here, I focus on three neurotransmitters/ neuromodulators and describe their actions (a) at the level of growth cone, especially on filopodia, which serve as sensors that allow growth cones to probe the environment they are traversing, and (b) on how neurotransmitters modulate neuronal electrical properties, which, in itself, have been shown to affect neurite extension. The goals of this dissertation are to investigate 1) the cholinergic modulation of neuronal activity and its effects on growth cone motility; 2) the excitatory modulation of neuronal excitability by nitric oxide (NO); and 3) the inhibitory modulation of neuronal activity by dopamine (DA). The work uses a well-established model system to investigate growth cone motility and neuronal activity: identified neurons from the pond snail Helisoma trivolvis studied in cell culture or in the intact ganglion in situ. The study of B5 neurons demonstrates that acetylcholine (ACh) induces filopodial elongation, which is mediated by opening of nicotinic ACh receptors, membrane depolarization, and elevation of intracellular Ca level in growth cones. This dissertation also shows that NO inhibits two types of Ca-activated K channels to depolarize the membrane potential of B19 neurons. Additionally, the study reveals that DA serves as an inhibitory neurotransmitter to hyperpolarize and silence the electrical activity of firing B5 neurons via a D2-like receptor/PLC/K channel pathway. Taken together, this dissertation elucidates novel cellular mechanisms through which neurotransmitters can regulate growth cone motility and neuronal electrical properties, further supporting evidence for potential roles of neurotransmitters in axon pathfinding and synaptic transmission in vivo.

Immune molecules, including complement proteins, C1q and C3, have emerged as critical mediators of synaptic refinement and plasticity. Complement proteins localize to synapses and refine the developing retinogeniculate system via C3-dependent microglial phagocytosis of synapses. Retinal ganglion cells (RGCs) express C1q, the initiating protein of the classical complement cascade, during retinogeniculate refinement; however, the signals controlling C1q expression and function remain elusive. RGCs grown in the presence of astrocytes significantly upregulated C1q compared to controls, implicating an astrocyte-derived factor in neuronal C1q expression. A major goal of my dissertation research was to identify the signals that regulate C1q expression and function in the developing visual system. In this study, I have identified transforming growth factor beta \((TGF-\beta)\), an astrocyte-secreted cytokine, as both necessary and sufficient for C1q expression in RGCs through an activity-dependent mechanism. Specific disruption of retinal \(TGF-\beta\) signaling resulted in a significant reduction in the deposition of C1q and downstream C3 at retinogeniculate synapses and significant synaptic refinement defects in the retinogeniculate system. Microglia engulfment of RGC inputs in the lateral geniculate nucleus (LGN) was also significantly reduced in retinal \(TGF\beta\)RII KOs, phenocopying the engulfment defects observed in C1q KOs, C3 KOs, and CR3 KOs. Interestingly, in C1q KOs and retinal \(TGF\beta\)RII KOs, microglia also failed to preferentially engulf less active inputs when retinal activity was manipulated, suggesting that retinal activity and \(TGF-\beta\) signaling cooperatively regulate complement mediated synaptic refinement. In support of this hypothesis, blocking spontaneous activity in RGC cultures significantly reduced C1q upregulation by \(TGF-\beta\). Moreover, manipulating spontaneous retinal activity in vivo modulated C1q expression levels in RGCs and C1q deposition in the LGN. Together these findings support a model in which retinal activity and \(TGF-\beta\) signaling control expression and local release of C1q in the LGN to regulate microglia-mediated, complement-dependent synaptic pruning. These results provide mechanistic insight into synaptic refinement and, potentially, pathological synapse loss which occurs in the early stages of neurodegenerative diseases concurrently with aberrant complement expression and reactive gliosis.

Axon degeneration is a selective elimination of axons, which plays a crucial role during development, injury, and maintenance of neuronal connections. The p75 neurotrophin receptor (NTR) is responsible for maintaining the specificity of neuronal connectivity in parts of the adult brain by inducing the degeneration of aberrantly growing axons into myelinated tracts. The objective of this study is to identify and characterize the signaling pathways used by p75NTR to mediate axon degeneration on myelin. Since p75NTR signals via JNK/Bax/caspase pathway to cause apoptosis, I asked whether this pathway might also be involved in axon degeneration. I observed that inhibition of JNK or Bax significantly decreased myelin-induced axonal degeneration, while depolarization of axons with potassium chloride prevented axonal degeneration on myelin. Together, these results suggest that p75NTR-dependent, myelin-mediated axon degeneration occurs via JNK/BAX signaling, and that neural activity is important for the prevention of myelin-induced axonal degeneration.

Axon degeneration is a selective elimination of axons, which plays a crucial role during development, injury, and maintenance of neuronal connections. The p75 neurotrophin receptor (NTR) is responsible for maintaining the specificity of neuronal connectivity in parts of the adult brain by inducing the degeneration of aberrantly growing axons into myelinated tracts. The objective of this study is to identify and characterize the signaling pathways used by p75NTR to mediate axon degeneration on myelin. Since p75NTR signals via JNK/Bax/caspase pathway to cause apoptosis, I asked whether this pathway might also be involved in axon degeneration. I observed that inhibition of JNK or Bax significantly decreased myelin-induced axonal degeneration, while depolarization of axons with potassium chloride prevented axonal degeneration on myelin. Together, these results suggest that p75NTR-dependent, myelin-mediated axon degeneration occurs via JNK/BAX signaling, and that neural activity is important for the prevention of myelin-induced axonal degeneration.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23757号 / 医博第4803号 / 新制||医||1056(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 岩田 想, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In addition to the acute pathology produced by traumatic brain injury, there are chronic alterations that occur after the trauma, including a depressed state of neuronal activity (Feeney, 1991). This study included a preclinical testing of a novel treatment strategy focusing on increasing neuronal activity during the chronic hypofunctional posttraumatic stage. The present investigation tested the effects of repeated post-injury aniracetam administration on cognitive performance in the Morris water maze (MWM) and on the GluR2 - immunoreactivity and protein expression by Western blot analysis in the hippocampus. The first study examined the optimal dose of aniracetam in the MWM task. Animals received aniracetam (25 mg/kg, 50 mg/kg) or vehicle once daily for fifteen days and on days 11-15 were tested in the MWM. The results indicated that injured aniracetam-treated rats had a significant improvement in MWM performance compared to injured saline-treated animals. When the drug was delayed for 11 days post-injury in the second experiment, its beneficial effects were still present, as injured aniracetam-treated rats performed significantly better that injured saline treated rats on the MWM task. In the third experiment, chronic daily aniracetam administration was terminated after 15 days immediately before MWM testing on days 16-20. The results indicated that termination of aniracetam did not enhance MWM performance as injured terminated aniracetam-treated rats did not have significant improvement over injured saline-treated rats. In the fourth study we investigated the mechanism of aniracetam's effects by examining the expression of the AMPA receptor GluR2 subunit, the only AMPA receptor subunit that is Ca++ impermeable. Using a monoclonal antibody selective for the GluR2 subunit, immunohistochemical results indicated that injured rats treated with aniracetam (50mg/kg for 15 days post-injury) had a slight reduction in the GluR2- IR. The fifth study investigated a change in the GluR2 protein expression in the hippocampus with a Western blot analysis. The results were consistent with the immunohistochemical study outcome as the injured vehicle and injured aniracetam treated animals showed a reduced protein expression in the hippocampus. The changes were not significantly different from the controls. The results of these experiments suggested that chronic aniracetam treatment significantly attenuated injury induced spatial memory deficits when administered continually during the hypofunctional posttraumatic stage and when the treatment was delayed for 11 days, but not when the treatment was terminated before the MWM testing. These effects suggest that the compound does not induce chronic receptor changes and has to be biologically active in an organism for it to exert its beneficial properties. Results from the present studies suggest that aniracetam may become a potential treatment option for brain injury induced cognitive deficits.

Sleep is well-established to become more superficial and fragmented as we age, with deficits in cognitive processing also commonly observed. While effects have been identified in both humans and mice (used in this thesis), there are important species differences in these findings and importantly, very little is known about the neural dynamics underlying these changes. By integrating several state-of-the-art approaches from putative single unit electrophysiological recordings to behavioural and pharmacological assessments, this thesis aimed to provide novel insights into the neural mechanisms involved in the age-dependent changes in sleep and cognition in mice. Firstly, this thesis investigated the neural activity underpinning the known global sleep changes that occur with ageing. Surprisingly, the majority of neuronal measures quantified in this study were resilient to the effects of ageing. Therefore the global sleep disruptions identified with ageing are unlikely to arise from changes in local cortical activity. Secondly, diazepam injection was found to suppress neural activity, in addition to previously reported effects on electroencephalography (EEG). Subtle differences in the effects of diazepam were identified across age groups, which may account for the variability seen in the efficacy of benzodiazepines in older individuals. Thirdly, ageing and sleep deprivation were found to have only a few effects on performance in a spatial learning task, the Morris water maze (MWM). Suggesting that spatial learning may be fairly resilient to the effects of ageing and sleep deprivation. Finally, this thesis presents preliminary analyses that showed mice were able to perform two novel paradigms of the visual discrimination task, suggesting their suitability in studying the link between ageing, sleep and cognition. Together the studies presented in this thesis provide insights into the differences between global and local mechanisms affected by ageing. Only by understanding local mechanisms will we be able improve on current treatments aimed at helping with the unwanted effects of healthy ageing, such as cognitive decline and sleep disruptions.