No description available.

Real-time tracking and object recognition is a large field with many possible applications. In this thesis we present a technical demo of a stereoscopic tracking system using artificial neural networks (ANN) and also an overview of the entire system, and its core functions. We have implemented a system able of tracking an object in real time at 60 frames per second. Using stereo matching we can extract the object coordinates in each camera, and calculate a distance estimate from the cameras to the object. The system is developed around the Xilinx ZC-706 evaluation board featuring a Zynq XC7Z045 SoC. Performance critical functions are implemented in the FPGA fabric. A dual-core ARM processor, integrated on the chip, is used for support and communication with an external PC. The system runs at moderate clock speeds to decrease power consumption and provide headroom for higher resolutions. A toolbox has been developed for prototyping and the aim has been to run the system with a one-push-button approach. The system can be taught to track any kind of object using an eight bit 32 × 16 pixel pattern generated by the user. The system is controlled over Ethernet from a regular workstation PC, which enables it to be very user-friendly.

Despite the importance of proper synaptogenesis in the CNS, the molecular mechanisms that regulate the formation and development of synapses remain poorly understood. Indeed, the mechanisms through which initial synaptic contacts are established and modified during synaptogenesis have not been fully determined and a precise understanding of these mechanisms may shed light on synaptic development, plasticity and many CNS developmental diseases. The development and formation of spiny synapses has been thought to occur via filopodia shortening followed by the recruitment of proper postsynaptic proteins, however the precise function of filopodia remains controversial. Thus the goal of this study was to investigate the dynamics of dendritic filopodia and determine their role in the development of synaptic contacts. We initially define and characterize short lipidated motifs that are sufficient to induce process outgrowth. Indeed, the palmitoylated protein motifs of GAP-43 and paralemmin are sufficient to induce filopodial extensions in heterologous cells and to increase the number of filopodia and dendritic branches in neurons. We showed that the morphological changes induced by these FIMs (filopodia inducing motifs) require on-going protein palmitoylation and are modulated by a specific GTPase, Cdc42, that regulates actin dynamics. We also show that their function is palmitoylation dependent and is dynamically regulated by reversible protein palmitoylation. Significantly, our work suggests a general role for those palmitoylated motifs in the development of structures important for synapse formation and maturation. We combined several approaches to monitor the formation and development of filopodia. We show that filopodia continuously explore the environment and probe for appropriate contacts with presynaptic partners. We find that shortly after establishing a contact with axons, filopodia induce the recruitment of presynaptic elements. Remarkably, we find that expression of acylated motifs or the constitutively active form of cdc-42 enhances filopodia number and motility, but reduces the recruitment of synaptophysin positive presynaptic elements and the probability of forming stable axo-dendritic contacts. We provide evidence for the rapid transformation of filopodia to spines within hours of imaging live neurons and reveal potential molecules that accelerate this process.

In this thesis, the transmission of spike trains in a neuron model is studied in order to obtain a better understanding of the role played by stochastic activity, i.e. uncorrelated spikes, in the auditory pathway. Fluctuations of the neuron membrane potential are given by a first-order stochastic differential equation, using a leaky integrate-and-fire model. In contrast to most previous studies the model has a finite number of synapses, and the usual diffusion approximation does not hold. / The input signal is modeled by spike trains with spiking times described by inhomogenous Poisson processes. The membrane potential is a shot noise process for which statistical properties are derived with a Gaussian approximation. The statistics of the output spike train are obtained by using the property that a pool of a large number of output spike trains can be modeled by an inhomogeneous Poisson process. It is shown that, under certain conditions, the addition of uncorrelated input spikes, i.e. noise, can enhance the transmission of periodic temporal information. This phenomenon, called stochastic resonance, is demonstrated analytically and supported by computer simulations. / Results are compared with those obtained from the traditional leaky integrate-and- fire neuron receiving a continuous waveform input. The shot-noise property of the membrane potential, which implies that its variance is de facto modulated by the input stimulus, is shown to enhance the phenomenon of stochastic resonance. Indeed, for a given average noise level, a modulated noise gives a higher output signal-to-noise ratio than an unmodulated noise with the same average amplitude. / The derivation is then extended to certain polyperiodic stimuli mimicking vowel sounds. The fact that the addition of uncorrelated input spikes can enhance the transmission of information is discussed in the context of cochlear implants. The results provide supportive evidence to the postulate that a cochlear implant speech coding strategy that elicits stochastic firing neural activity might benefit the user.

Recent studies have uncovered de novo mutations of the gene encoding the chromatin remodelling protein Snf2l in patients with schizophrenia, Rett-like syndrome and intellectual disability. Snf2l and its closely related protein, Snf2h, play a critical role in embryonic and post-natal brain development. Murine models lacking functional Snf2h or Snf2l point to complementary activities of these remodelers; Snf2h cKO mice present with a significantly reduced cerebellum, while Snf2l Ex6DEL (exon 6 deleted) cerebella are larger than their wild-type counterparts. Granule neuron progenitors (GNPs) isolated from Ex6DEL cerebella display delayed cell cycle exit and hindered terminal differentiation compared to wild-type controls. Moreover, loss of Snf2l activity results in widespread transcriptome shifts which underlie the Ex6DEL GNP differentiation phenotype. In particular, key transcription factors are differentially expressed without Snf2l remodelling activity. We confirm that ERK pathway activation is misregulated in Ex6DEL GNPs, possibly in response to elevated fibroblast growth factor 8 (Fgf8) expression in these cultures. We find that Snf2l activity maintains the chromatin landscape throughout GNP differentiation, as Ex6DEL cultures have a global increase in chromatin accessibility. We suggest that Snf2l-mediated chromatin condensation is responsible for proper regulation of gene expression programs in GNP differentiation.

Hypothermia is potently neuroprotective, but the molecular basis of this effect remains obscure and the practical challenges of cooling have restricted its clinical use. This thesis was borne on the premise that considerable therapeutic potential may lie in a deeper understanding of the neuronal physiology of cooling. Rodent studies indicate that hypothermia can elicit preconditioning wherein a subtoxic stress confers resistance to an otherwise lethal injury. This cooling-induced tolerance requires de novo protein synthesis – a fundamental arm of the cold-shock response, for which data in human neurons is lacking. Since cooling protects the human neonatal brain, experiments herein address the molecular effects of clinicallyrelevant cooling using functional, maturationally-comparable cortical neurons differentiated from human pluripotent stem cells (hCNs). Several core hypothermic phenomena are explored, with particular scrutiny of neuronal tau, since this protein is modified extensively in brains that are resistant to injury. Mild-to-moderate hypothermia produces an archetypal cold-shock response in hCNs and protects them from oxidative and excitotoxic stress. Principal features of human cortical tau development are recapitulated during hCN differentiation, and subsequently reversed by cooling, returning tau transcriptionally and post-translationally to an earlier foetallike state. These findings provide the first evidence of cold-stress-mediated ontogenic reversal in human neurons. Furthermore, neuroprotective hypothermia induces mild endoplasmic reticulum (ER) stress in hCNs, with subsequent activation of the unfolded protein response (UPR). Reciprocal modulation of both tau phosphorylation and the ER-UPR cascade suggests that cold-induced hyperphosphorylation of tau and ER-hormesis (preconditioning) represent significant components of hypothermic neuroprotection. Cooling thus modifies proteostatic pathways in a manner that supports neuronal viability. Historically, hypothermic preconditioning has been limited to the acute injury setting, and tau hyperphosphorylation is an established hallmark of chronic neural demise. More recently however, preconditioning has been proposed as a target for neurodegenerative disease and neuroprotective roles of phospho-tau have emerged. To date, hypothermia has protected hCNs against oxidative, excitotoxic and ER stress, all of which have been implicated in traumatic as well as degenerative processes. This ‘cross-tolerance’ effect places exponential value on the molecular neurobiology of cooling, with the potential to extract multiple therapeutic targets for an unmet need.

Despite the importance of proper synaptogenesis in the CNS, the molecular mechanisms that regulate the formation and development of synapses remain poorly understood. Indeed, the mechanisms through which initial synaptic contacts are established and modified during synaptogenesis have not been fully determined and a precise understanding of these mechanisms may shed light on synaptic development, plasticity and many CNS developmental diseases. The development and formation of spiny synapses has been thought to occur via filopodia shortening followed by the recruitment of proper postsynaptic proteins, however the precise function of filopodia remains controversial. Thus the goal of this study was to investigate the dynamics of dendritic filopodia and determine their role in the development of synaptic contacts. We initially define and characterize short lipidated motifs that are sufficient to induce process outgrowth. Indeed, the palmitoylated protein motifs of GAP-43 and paralemmin are sufficient to induce filopodial extensions in heterologous cells and to increase the number of filopodia and dendritic branches in neurons. We showed that the morphological changes induced by these FIMs (filopodia inducing motifs) require on-going protein palmitoylation and are modulated by a specific GTPase, Cdc42, that regulates actin dynamics. We also show that their function is palmitoylation dependent and is dynamically regulated by reversible protein palmitoylation. Significantly, our work suggests a general role for those palmitoylated motifs in the development of structures important for synapse formation and maturation. We combined several approaches to monitor the formation and development of filopodia. We show that filopodia continuously explore the environment and probe for appropriate contacts with presynaptic partners. We find that shortly after establishing a contact with axons, filopodia induce the recruitment of presynaptic elements. Remarkably, we find that expression of acylated motifs or the constitutively active form of cdc-42 enhances filopodia number and motility, but reduces the recruitment of synaptophysin positive presynaptic elements and the probability of forming stable axo-dendritic contacts. We provide evidence for the rapid transformation of filopodia to spines within hours of imaging live neurons and reveal potential molecules that accelerate this process. / Medicine, Faculty of / Graduate

Cigarette smoking during pregnancy contributes to the development of neurological health problems in offspring. As a result, public health organizations are recommending NRT to pregnant women to wean them off tobacco. If nicotine itself is injurious to the developing brain, then nicotine substitution may not eliminate the deleterious health outcomes of maternal smoking. In studies of cognitive decline, estradiol elicits a neuroprotective effect through ER activation. However, the underlying mechanism remains unclear. Evidence suggests that estrogen-mediated neuroprotection is activated through glial cell interaction, mitigating inflammation and protecting neurons critical for learning and memory. If NRT antagonizes these cellular targets, it may put individuals at risk for future cognitive impairments. Randomly assigned nulliparous female Wistar rats were injected subcutaneously with 1 mg/kg/day of nicotine bitartrate or saline for 2 weeks before mating until weaning (PND 21). Pups (saline n=6 and nicotine, n=6) were sacrificed at 26 weeks of age and the hippocampal formation was processed for Nissl and immunohistochemical staining for GFAP and ERα. Gestational exposure to nicotine only produced a significant increase in the expression of ERα in the DG of the hippocampus. While additional research is needed, these findings suggest that NRT might indeed interfere with proper brain development, making offspring increasingly susceptible to long-term adverse health effects. Le tabac fumé pendant la grossesse affecte de manière importante le développement neurologique de la progéniture, y compris à long terme. C’est pourquoi, les autorités de la santé publique recommandent les substituts nicotiniques comme soutien au sevrage tabagique chez les femmes enceintes. Si le danger se situe dans la nicotine de la cigarette, alors les produits de substitution nicotiniques risquent également d’interférer avec le développement cérébral. De nombreuses données expérimentales convergent pour attribuer un rôle protecteur à l’oestradiol sur le fonctionnement cognitif. Par contre, le mécanisme sous-jacent est inconnu. Il se peut que l’oestradiol arrive à neutraliser la réaction inflammatoire provoquée par les cellules gliales, amoindrissant la détérioration des neurones impliqués au niveau de la mémoire. Ainsi, une perturbation de ce mécanisme par la nicotine pourrait engendrer une détérioration progressive des fonctions cognitives. Des rats femelles Wistars nullipares assignées de façon alléatoire à un groupe ont reçu soit une injection sous-cutanée de 1mg/kg/jour de nicotine bitartrate ou de saline, 2 semaines avant l’accouplement jusqu'au sevrage au jour 21 postnatal. A 26 semaines, les ratons furent sacrifiés (saline n=6 et nicotine, n=6) et une analyse du Nissl et immunohistochimique de GFAP et ERα furent réalisées sur les formations hippocampiques. L’exposition prénatale à la nicotine a seulement augmenté significativement l’expression de ERα dans le GD de l’hippocampe. Alors que des études plus approfondies sont nécessaires, ces résultats suggèrent que les substituts nicotiniques affectent le développement neurologique périnatal, ce qui risque d’entrainer des répercussions a long terme sur la santé.

Autism spectrum disorders (ASDs) are a heterogeneous class of neurodevelopmental disorders that share the three core behavioral symptoms of impaired social interactions, communication deficits, and restrictive and repetitive behaviors. Our previous studies identified the novel X-linked ASD gene NEXMIF (KIAA2022, KIDLIA). Mutations in NEXMIF leading to loss of its protein product are responsible for the development of autistic features and intellectual disability in humans. However, the role NEXMIF plays in brain development and ASD remains largely unknown. Therefore, I investigated the behavioral impairments and cellular and molecular dysregulation that result from loss of NEXMIF in a transgenic mouse model. I found that male NEXMIF-/y hemizygous knockout (KO) mice replicate the behavioral alterations reported in affected humans and that cultured neurons from NEXMIF-/y KO brains show a significant decrease in neurite outgrowth, synaptic protein expression, and spine and synapse density. Loss of NEXMIF in cultured neurons also leads to altered expression of many genes including several involved in synaptic development and function. Reintroduction of some of the downregulated genes in cultured neurons rescued the decreased spine density and synaptic AMPAR levels observed from loss of NEXMIF. Several clinical reports have indicated that in females, haploinsufficiency of the X-linked NEXMIF gene causes symptoms similar to those observed in males lacking NEXMIF. Therefore, I examined the behavioral and molecular phenotypes in a transgenic mouse model of NEXMIF haploinsufficiency, female NEXMIF+/- mice. These animals displayed ASD-like behaviors, including impaired social interactions, repetitive self-grooming, and memory deficits. NEXMIF haploinsufficiency results in mosaic expression of the protein, resulting in two populations of neurons in the brain, those that express NEXMIF and those that do not. Interestingly, I found that both types of neurons demonstrated impairments in dendritic outgrowth, synaptic density, and the expression of important synaptic proteins. Together, these findings provide new insights into the cellular and molecular mechanisms of NEXMIF-dependent ASD and the role of NEXMIF in neurodevelopment, in both males and females.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13163号 / 論医博第2150号 / 新制||医||1029(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 林 康紀, 教授 渡邉 大, 教授 高橋 淳 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM