The correct polarity of a neuron underlies its ability to integrate precise circuitries in the nervous system. The goal of my thesis was to investigate the pathways that establish and maintain neuron polarity/orientation in vivo. To accomplish this, I used bipolar VC4/5 motor neurons, which innervate the C. elegans egg-laying musculature, as a model system. Vulval proximal VC4/5 neurons extend axons in the left-right (LR) orientation, around the vulva; whereas vulval distal VC1-3,6 neurons extend axons along the anterior-posterior (AP) axis. A previous study showed that vang-1, a core planar cell polarity (PCP) gene, suppresses AP axon growth in VC4/5 neurons. In order to identify new components of this pathway we performed genetic screens for mutants with abnormal VC4/5 polarity/morphology. We isolated and mapped alleles of farnesyl transferase b (fntb-1) and of core PCP genes, prickle- 1 (prkl-1) and dishevelled-1 (dsh-1); all of which display tripolar VC4/5 neurons, similar to vang-1 lof. In prkl-1 and dsh-1 mutants, primary LR and ectopic AP VC4/5 axons are born simultaneously, suggesting an early role in establishing polarity. In addition, prkl-1 and dsh-1 act persistently to maintain neuron morphology/orientation. Genetic analysis of double mutants suggests that prkl-1 interacts with vang-1 in a common PCP pathway to prevent AP axon growth, while dsh-1 also acts in a parallel pathway. Furthermore, prkl-1 functions cell autonomously in neurons, whereas dsh-1 acts both cell autonomously and cell nonautonomously in epithelial cells. Notably, prkl-1 overexpression results in unipolar VC4/5 neurons, in a dose-dependent manner. In contrast, dsh-1 overexpression in VC4/5 neurons results in a lof phenotype, similar to vang-1 lof and overexpression phenotype. Remarkably, prkl-1 overexpression restores normal VC4/5 polarity in dsh-1 and vang-1 mutants, which is suggestive of a downstream role for prkl-1. Both PRKL-1 and DSH-1 are expressed in iii uniformly distributed puncta at the plasma membrane of VC4/5, similar to VANG-1; suggesting that their asymmetric distribution is not critical for neuron polarity. Furthermore, we found that the vulva epithelium induces prkl-1 expression in VC4/5; indicating a functional relationship between the egg-laying organ and neuron morphology. Moreover, a structure-function analysis of PRKL-1 revealed that the conserved PET domain and the Cterminal region are crucial to prevent AP axon growth, whereas the three LIM domains are dispensable for this role. In addition, we showed that dsh-1 also regulates the morphology of AP-oriented PDE neurons. dsh-1 promotes the formation of PDE posterior axons, contrary to its function in VC5 neurons; which indicates a context-dependent role for dsh-1 in neuronal polarity. Altogether, this thesis implicates the PCP signalling pathway in a previously unknown role, in establishing and maintaining neuronal polarity, by controlling AP axon growth in response to organ-derived polarizing cues.

Synapses are specialized sub-cellular junctions that transmit signals between neurons and their targets. In Caenorhabditis elegans (C. elegans) the F-box protein FSN-1 and the PHR family member RPM-1 form the SCFFSN-1 E3 ubiquitin ligase, which plays an important role in regulating synaptic growth factors. This SCF complex is evolutionarily conserved across species, and regulates many cellular processes including axon outgrowth, apoptosis and synaptogenesis. This thesis focuses on identifying targets of SCFFSN-1 that contribute to synaptogenesis. Forward genetics was employed to screens and isolate mutants that exhibit genetic interactions with fsn-1. I have identified an allele of the MAPK pmk-3(hp246) and three alleles of the MAPKKK dlk-1(hp180, hp192, hp195) that suppress fsn-1 defects. In addition, I have isolated five fsn-1 suppressing alleles and evidence suggests that these suppressors are likely novel fsn-1 suppressors.

Synapses are specialized sub-cellular junctions that transmit signals between neurons and their targets. In Caenorhabditis elegans (C. elegans) the F-box protein FSN-1 and the PHR family member RPM-1 form the SCFFSN-1 E3 ubiquitin ligase, which plays an important role in regulating synaptic growth factors. This SCF complex is evolutionarily conserved across species, and regulates many cellular processes including axon outgrowth, apoptosis and synaptogenesis. This thesis focuses on identifying targets of SCFFSN-1 that contribute to synaptogenesis. Forward genetics was employed to screens and isolate mutants that exhibit genetic interactions with fsn-1. I have identified an allele of the MAPK pmk-3(hp246) and three alleles of the MAPKKK dlk-1(hp180, hp192, hp195) that suppress fsn-1 defects. In addition, I have isolated five fsn-1 suppressing alleles and evidence suggests that these suppressors are likely novel fsn-1 suppressors.

This thesis focuses on the regulation of the ubiquitous second messenger Ca2+ in neuronal physiology and disease. Ca2+ signalling in neurons is regulated by ion channels located in the plasma membrane, as well as in the endoplasmic reticulum (ER) and mitochondrial membranes. Ca2+ signalling is essential for numerous cellular processes, including neuronal excitability, neurotransmitter release, synaptic plasticity, and induction of cell death. Age-related disruptions in Ca2+ signalling may contribute to decline of cognitive function and motor control associated with aging. Furthermore, disruption in neuronal Ca2+ signalling is implicated in several neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and Amyotrophic Lateral Sclerosis (ALS). In this thesis, I studied neuronal Ca2+ signalling and how it is affected in neurodegenerative disease. First, I examined the role of the ER Ca2+ binding protein Calreticulin (CRT) in AD. CRT is involved in regulation of ER Ca2+ signalling and modulation of susceptibility to cell death. I found that there was an increase in the expression of CRT in in vitro and in vivo models of AD. However, increased levels of CRT did not alter susceptibility of neuronal cells to death induced by AD-related stressors. Second, I examined the role of X-Linked Inhibitor of Apoptosis Protein (XIAP) in the modulation of neuronal Ca2+ signalling. I found that overexpression of XIAP in neuronal cells modified Ca2+ signalling by decreasing Ca2+ flux through multiple plasma membrane and ER channels. These effects appear to be independent of caspase inhibition, which is one of the ways that XIAP can inhibit apoptosis. Third, I examined a compound found in green tea, L-theanine, a glutamate receptor antagonist that is protective in models of excitotoxic neuronal injury. I found that 24 hour L-theanine treatment reduces the amount of Ca2+ released from neuronal intracellular stores in response to both glutamate stimulation and passive leak through ER channels. An acute 30 minute L-theanine treatment had similar effects. In conclusion, these observations further the understanding of the regulation of Ca2+ signalling in neurons and may lead to novel therapeutic strategies in neurodegenerative disease. / October 2008

No description available.

Multistability is a fundamental attribute of the dynamics of neuronal systems under normal and pathological conditions. The mechanism of bistability of bursting and silence is not well understood and to our knowledge has not been experimentally recorded in single neurons. We considered four models. Two of them described the dynamics of a leech heart interneuron: the canonical model and a low-dimensional model. The other two models described mammalian pacemakers from the respiratory center. We investigated the low-dimensional model and identified six different types of multistability of dynamical regimes. We described six generic mechanisms underlying the co-existence of oscillatory and silent regimes. The mechanisms are based either on a saddle equilibrium or a saddle periodic orbit. The stable manifold of the saddle equilibrium or the saddle orbit sets the threshold between the regimes. In the two models of the leech interneuron the range of the controlling parameters supporting the co-existence of bursting and silence is limited by the Andronov-Hopf and homoclinic bifurcations (Malashchenko, Master Thesis 2007). The bistability was found in a narrow range of the leak currents' parameters. Here, we introduced a propensity index to bistability as the width of the range on a bifurcation diagram; we investigated how the propensity index was affected by modifications of the ionic currents, and found that conductances of only two currents substantially affected the index. The increase of the conductance of the hyperpolarization-activated current, Ih, and the reduction of the fast Ca2+ current, ICaF, notably increased the propensity index. These findings define modulatory conditions under which we suggest the bistability of bursting and silence could be experimentally revealed in leech heart interneurons. We hypothesize that this mechanism could be commonly found in a large variety of neuronal models. We applied our techniques to models of vertebrate neurons controlling respiratory rhythm, which represent two types of inspiratory pacemakers of the Pre-Bӧtzinger Complex. We showed that both types of neurons could exhibit bistability of bursting and silence in accordance with the mechanism which we described.

The objective of this research is to develop a three-dimensional (3-D) microfluidic/ electronic interface system for sustaining and monitoring 3-D neuronal networks. This research work is divided into two parts. One is the development of a 3-D multi-electrode array (MEA) with integrated microfluidic channels. The other is a microneedle array with embedded microelectrodes and microfluidic channels. The 3-D MEA is composed of three elements that are essential for the development and monitoring of 3-D cultures of neurons. These components consist of scaffolds for cellular growth and structural stability, microfluidic channels for cell maintenance and chemical stimulation, and electrodes for electrical stimulation and recording. Two kinds of scaffold structures have been fabricated. The first scaffolding scheme employs a double exposure technique that embeds SU-8 towers into an SU-8 substrate. The second scaffolding mechanism introduces interconnects between towers for the purpose of mechanically supporting 3-D cell cultures and facilitating 3-D synaptic connections. Microfluidic channels are combined for fine control of the cellular microenvironment by means of diffusive and convective fluidic processes. Hollow towers with three-layer side ports were developed by using double exposure techniques and excimer laser ablation. The electrodes are combined into an integrated system that is capable of monitoring electrical activities and the cellular impedances of neurons which are attached to the electrodes. The second part of this research is to fabricate a microneedle array for monitoring brain slices, which will directly detect electrical signals from living brain slices. Although the microneedle array is targeting different 3-D neuronal networks, it also has three components and the fabrication steps are the same as those for the 3-D MEA. To generate the sharp tip, isotropic reactive ion etching (RIE) is performed on tapered SU-8 towers. High aspect ratio tower structures can be effectively generated with SU-8 and tapered shapes are created by backside exposure. The resulting systems will enable a new field of neurobiological research, in which the collective properties of 3-D neuronal circuits can be observed and manipulated with unprecedented detail and precision, and at a level of control not possible in living animals.

The pre-Botzinger complex (pBC) is a sub-circuit of the respiratory central pattern generator. The pBC is required for eupnea and is contained in a transverse slice of the ventrolateral medulla. In the slice, pBC cells are responsible for generating the respiratory rhythm, and hypoglossal motoneurons (HMs) are responsible for transmitting the rhythm out of the brainstem to the muscles. Understanding how the transverse slice rhythm is generated and transmitted is a first step in understanding how this process occurs in vivo. To understand this network, we developed ionic current models of the individual network components and explored how the various ion channels affect single-cell firing characteristics and network dynamics. First, we used the considerable amounts of experimental data from neonatal HMs to develop an HM model. The model was used to explore the roles of ion channels in shaping the complex dynamics of the neonatal HM action potential (AP) and to investigate the age-dependent changes in HMs. We used a genetic algorithm to optimize the HM model to more closely fit experimental measures of AP shape. A comparison of feature-based and template-based fitness functions revealed that a feature-based fitness function performs best when optimizing the HM model to fit characteristics of the neonatal HM AP. Next, we used our existing pBC models to understand how different ionic currents affect rhythmogenesis in the pBC. Our results indicate that intrinsic bursters increase the robustness of rhythm generation in the pBC. Finally, we developed an improved pBC neuron model and explored how various ion channels affect bursting dynamics at the single-cell level. The HM and pBC models developed in this study will be used in future network models of the transverse slice.

Sexual differentiation is divided into gonadal sexual differentiation and brain sexual differentiation in the teleosts. Gonadal sexual differentiation is regulated by brain sexual differentiation. Brain sexual differentiation is resulted from the neural development, which lead to the sexual dimorphism of both structure and functions of brain. The neural development is influenced by the genetic factors and the external environmental factors. The lower temperature induces a higher proportion of female while the elevated temperature induces a higher proportion of male in tilapia. In addition, there is sexual difference in effects of temperature on the activity of brain neurochemicals. Water temperature plays an important role on the development of central neurotransmitter systems and sexual differentiation during the developing period. In the present study, the primary neural culture cloned from the female and male tilapia¡AOreochromis mossambicus was used. The difference of the physiological characteristics between the neural cells derived from the females and males, were investigated. These results show that the elevated temperature has an effect to enhance the proliferation in both primary neural culture of females and males.

(運動・神経生理)