Intellectual disability (ID) is a common phenotype of brain-development disorders and is heterogeneous in etiology with numerous genetic causes. PAK3 is one gene with multiple mutations causing ID. Affected individuals have microcephaly, and other brain-structure defects have been reported. Additionally, PAK3 is in a genetic network with eighteen other genes whose mutations cause ID, suggesting the molecular mechanisms by which PAK3 regulates of cognitive function may be shared by other genetic ID disorders. Studies in rodent models have shown that the orthologs of PAK3 are important for regulating dendrite spine morphology and postnatal brain size. In Drosophila melanogaster, the morphological processes of oogenesis, dorsal closure during embryogenesis, and salivary gland-lumen formation require Pak, the Drosophila ortholog of PAK3. Additionally, Pak is important for development of the subsynaptic reticulum of the neuromuscular junction, sensory axon pathfinding and terminal arborization in the Drosophila central nervous system (CNS). However, the role of Pak in mushroom body (MB) structure and intrinsic neurite arbor morphogenesis, as well as details of the underlying cellular and molecular mechanisms are unknown. To address this gap, I used Drosophila models of PAK3 gene mutations, Pak, and a combination of immunostaining, primary cell culture, and genetic interaction studies to elucidate these mechanisms. I performed a detailed characterization of the previously reported adult Pak phenotypes of decreased survival as well as leg and wing morphology. I found that decreased survival is a low-penetrance phenotype that is enhanced by chromosomes from the same mutagenesis. Defects of the adult wing include folding and misalignment between the layers, blisters, and missing or partial cross veins. The Pak-mutant legs are short and often misdirected in the pupal case with morphological defects in the shape of the leg segments themselves. The mushroom bodies are important insect learning and memory brain structures whose lobes are composed of axon bundles with individual axons bifurcating to form the α and β lobes. Mutations in Pak cause defects in the length, thickness, and direction of the MB α and β lobes. These defects increase in severity during metamorphosis, when neurogenesis and differentiation of these structures occur, suggesting that Pak stabilizes the branches of the α/β mushroom body neurons. Pak-mutant cultured neurons have reduced neurite arbor size with defects in neurite caliber. Initial outgrowth was normal, followed by a decrease in neurite branch number, again supporting the role of Pak in neurite-branch stability. There are defects in the cytoskeleton in growth cones at six hours post-plating as well as in neurons after three days in vitro. The Pak-mutant phenotype severity depends on the phosphorylation status of myosin regulatory light chain, supporting the mechanistic hypothesis that Pak regulates neurite-branch stability by inhibiting myosin light chain kinase. The neuronal phenotype of decreased branch stability suggests a mechanism of excessive retraction as the cellular pathogenesis underlying PAK3 mutation-associated brain disorders. I used western blotting to characterize the protein products of four nonsense mutations in Drosophila Pak to interpret genotype-phenotype relationships. Each allele has molecularly unique consequences: Pak¹¹, stop-codon read through and truncated protein; Pak¹⁶, no read through, but truncated protein; Pak⁶, read through with no truncated protein; Pak ¹⁴, neither readthrough nor truncated protein. Truncated proteins produced by Pak¹¹ and Pak¹⁶ alleles retained partial function for survival, wing blistering, leg morphology, and neurite length. Conversely, truncated protein increased the severity of the mushroom body defects. Truncated proteins have no effect on neuron branch number, wing folding, or vein defects. Together, these results demonstrate a role of Pak in regulating epithelial morphology, brain structure, and neurite arbor size and complexity. These closely resemble features of the human disorder, providing evidence that this is a good genetic model for this cause of ID.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative condition that affects corticospinal and spinal motor neurons and leads to death within 30 months of symptom onset in half of all cases. It remains incurable and treatment is supportive. The genetic and molecular understanding of ALS has gone through a rapid expansion in recent years, notably with the discoveries of TDP-43, a heterogeneous ribonucleoprotein as a major component of neuronal inclusions in ALS, as well as the discovery of the C9orf72 hexanucleotide expansion as the most common genetic cause of this disease. This first part of this thesis addresses the question of which of the various pathological hallmarks of the C9orf72 Hexanucleotide Repeat Expansion (HRE) in autopsy material correlates best with the clinical presentation. The main finding is that TDP-43 distribution, rather than C9orf72 RNA foci or dipeptide aggregation in the brain, corresponds best with the areas relevant to the clinical subtype of ALS-FTD. Subsequently the role of TDP-43 was investigated in induced pluripotent stem cell derived motor neurons, and no evidence of the hallmarks of TDP-43 dysfunction, were seen in this model of the disease. No mislocalisation is found on immunofluorescence, and biochemical analysis shows no differences in insoluble species between the patient and control cell lines. In the final section, RNA sequencing was used to study the transcriptome of a BAC transgenic mouse carrying a human M337V transgene expressed at low levels, to identify early presymptomatic differences in gene expression. Interestingly, no changes were found in genes known to be associated with ALS through mutations, and the constitutive nuclear functions of TDP-43 in the regulation of splicing was maintained, prior to the emergence of a clinical phenotype in the mouse. This favours a gain of function mechanism for TDP-43 mutations in ALS.

Examining the effect of repeating stimuli on brain activity is important for theories of perception, learning and memory. Functional magnetic resonance imaging (fMRI) is a non-invasive way to examine repetition-related effects in the human brain. However the Blood-Oxygenation Level-Dependent (BOLD) signal measured by fMRI is far removed from the electrical activity recorded from single cells in animal studies of repetition effects. Despite that, there have been many claims about the neural mechanisms associated with fMRI repetition effects. However, none of these claims has adequately considered the temporal and spatial resolution limitations of fMRI. In this thesis, I tackle these limitations by combining simulations and modelling in order to infer repetition-related changes at the neural level. I start by considering temporal limitations in terms of the various types of general linear model (GLM) that have used to deconvolve single-trial BOLD estimates. Through simulations, I demonstrate that different GLMs are best depending on the relative size of trial-variance versus scan-variance, and the coherence of those variabilities across voxels. To address the spatial limitations, I identify six univariate and multivariate properties of repetition effects measured by event-related fMRI in regions of interest (ROI), including how repetition affects the ability to classify two classes of stimuli. To link these properties to underlying neural mechanisms, I create twelve models, inspired by single-cell studies. Using a grid search across model parameters, I find that only one model (“local scaling”) can account for all six fMRI properties simultaneously. I then validate this result on an independent dataset that involves a different stimulus set, protocol and ROI. Finally, I investigate classification of initial versus repeated presentations, regardless of the stimulus class. This work provides a better understanding of the neural correlates of stimulus repetition effects, as well as illustrating the importance of formal modelling.

Visual perception in mammals is made possible by the visual system and the visual cortex. However, precisely how visual information is coded in the brain and how training can improve this encoding is unclear. The ability to see and process visual information is not an innate property of the visual cortex. Instead, it is learnt from exposure to visual stimuli. We first considered how visual perception is learnt, by studying the perceptual learning of contrast discrimination in macaques. We investigated how changes in population activity in the visual cortices V1 and V4 correlate with the changes in behavioural response during training on this task. Our results indicate that changes in the learnt neural and behavioural responses are directed toward optimising the performance on the training task, rather than a general improvement in perception of the presented stimulus type. We report that the most informative signal about the contrast of the stimulus within V1 and V4 is the transient stimulus-onset response in V1, 50 ms after the stimulus presentation begins. However, this signal does not become more informative with training, suggesting it is an innate and untrainable property of the system, on these timescales at least. Using a linear decoder to classify the stimulus based on the population activity, we find that information in the V4 population is closely related to the information available to the higher cortical regions involved with decision making, since the performance of the decoder is similar to the performance of the animal throughout training. These findings suggest that training the subject on this task directs V4 to improve its read out of contrast information contained in V1, and cortical regions responsible for decision making use this to improve the performance with training. The structure of noise correlations between the recorded neurons changes with training, but this does not appear to cause the increase in behavioural performance. Furthermore, our results suggest there is feedback of information about the stimulus into the visual cortex after 300 ms of stimulus presentation, which may be related to the high-level percept of the stimulus within the brain. After training on the task, but not before, information about the stimulus persists in the activity of both V1 and V4 at least 400 ms after the stimulus is removed. In the second part, we explore how information is distributed across the anatomical layers of the visual cortex. Cortical oscillations in the local field potential (LFP) and current source density (CSD) within V1, driven by population-level activity, are known to contain information about visual stimulation. However the purpose of these oscillations, the sites where they originate, and what properties of the stimulus is encoded within them is still unknown. By recording the LFP at multiple recording sites along the cortical depth of macaque V1 during presentation of a natural movie stimulus, we investigated the structure of visual information encoded in cortical oscillations. We found that despite a homogeneous distribution of the power of oscillations across the cortical depth, information was compartmentalised into the oscillations of the 4 Hz to 16 Hz range at the granular (G, layer 4) depths and the 60Hz to 170Hz range at the supragranular (SG, layers 1–3) depths, the latter of which is redundant with the population-level firing rate. These two frequency ranges contain independent information about the stimulus, which we identify as related to two spatiotemporal aspects of the visual stimulus. Oscillations in the visual cortex with frequencies < 40 Hz contain information about fast changes in low spatial frequency. Frequencies > 40 Hz and multi-unit firing rates contain information about properties of the stimulus related to changes, both slow and fast, at finer-grained spatial scales. The spatiotemporal domains encoded in each are complementary. In particular, both the power and phase of oscillations in the 7 Hz to 20Hz range contain information about scene transitions in the presented movie stimulus. Such changes in the stimulus are similar to saccades in natural behaviour, and this may be indicative of predictive coding within the cortex.

The adoption and subsequent retention of distinct cellular fates upon cell division is a critical phenomenon in the development of multicellular organisms. A well-studied example of this process is stem cell divisions; stem cells must possess the capacity to self-renew in order to maintain a stem cell population, as well as to generate differentiated daughters for tissue growth and repair. Drosophila neuroblasts are the neural stem cells of the central nervous system and have emerged as an important model for stem cell divisions and the genetic control of daughter cell identities. Neuroblasts divide asymmetrically to generate daughters with distinct fates; one retains a neuroblast identity and the other, a ganglion mother cell, divides only once more to generate differentiated neurons and glia. Perturbing the asymmetry of neuroblast divisions can result in the failure to self-renew and the loss of the neural stem cell population; alternatively, ectopic self-renewal can occur, resulting in excessive neuroblast proliferation and tumorigenesis. Several genetic lesions have been characterized which cause extensive ectopic self-renewal, resulting in brains composed of neuroblasts at the expense of differentiated cells. This contrasts with wild type brains, which are composed mostly of differentiated cells and only a small pool of neuroblasts. We made use of these mutants by performing a series of microarray experiments comparing mutant brains (consisting mostly of neuroblasts) to wild type brains (which are mostly neurons). Using this approach, we generated lists of over 1000 putatively neuroblast-expressed genes and over 1000 neuronal genes; in addition, we were able to compare the transcriptional output of different mutants to infer the neuroblast subtype specificity of some of the transcripts. Finally, we verified the self-renewal function of a subset of the neuroblast genes using an RNAi-based screen, resulting in the identification of 84 putative self-renewal regulators. We went on to show that one of these genes, midlife crisis (mammals: RNF113a), is a well-conserved RNA splicing regulator which is required in postmitotic neurons for the maintenance of their differentiated state. Our data suggest that the mammalian ortholog performs the same function, implicating RNF113a as an important regulator of neuronal differentiation in humans.

Old-age hippocampal sclerosis (HS), characterised by severe neuron loss in hippocampal CA1, is a poorly understood cause of dementia. At present no objective pathological HS criteria exist. In life HS is commonly diagnosed as Alzheimer's disease. HS aetiology is unclear, although it has been associated with both ischaemia and TAR-DNA-binding protein-43 (TDP-43)-related neurodegeneration. Variations in genes GRN, TMEM106B and ABCC9 are proposed as HS risk factors. The aim of this thesis was to investigate epidemiological, clinical, pathological and genetic characteristics of HS in older European populations. 976 brains donated for the Cambridge City over-75s Cohort, the Cognitive Function and Ageing Study and the Finnish Vantaa 85+ study were available for evaluation -including bilateral hippocampi from 302 individuals. A protocol capturing the extent and severity of hippocampal neuron loss was developed, establishing objective HS diagnosis criteria and allowing observation of distinct neuron loss patterns associated with ischaemia and neurodegeneration. 71 HS cases (overall prevalence: 7.3%) were identified. HS was significantly associated with an advanced age at death as well as dementia at the end of life. Neuropsychological and cardiovascular characteristics were similar between HS and AD, except for a longer duration of dementia and more disability in HS. HS was not associated with neurofibrillary tangles, amyloid plaques, or vascular pathologies, but all HS cases evaluated for TDP-43 showed neuronal inclusions in the hippocampal dentate and a high frequency of other glial, neuronal and neurite TDP-43 pathologies. GRN and TMEM106B but not ABCC9 variations were linked to HS. A moderating effect of TDP-43 on this association was detected. HS presented pathologically similarly to frontotemporal dementia cases with TDP-43 (FTLD-TDP) caused by mutations in GRN, but differed from other FTLD-TDP subtypes. Results of this thesis reveal the importance of HS in the oldest old in the population, the key role of TDP-43, as well as providing robust methods to capture HS characteristics for an area that has been under-researched but is clearly vital to understanding dementia in the oldest old.

abstract: The Erk/MAPK pathway plays a major role in cell growth, differentiation, and survival. Genetic mutations that cause dysregulation in this pathway can result in the development of Rasopathies, a group of several different syndromes including Noonan Syndrome, Costello Syndrome, and Neurofibromatosis Type-1. Since these mutations are germline and affect all cell types it is hard to differentiate the role that Erk/MAPK plays in each cell type. Previous research has shown that individual cell types utilize the Erk/MAPK pathway in different ways. For example, the morphological development of lower motor neuron axonal projections is Erk/MAPK-independent during embryogenesis, while nociceptive neuron projections require Erk/MAPK to innervate epidermal targets. Here, we tested whether Erk/MAPK played a role in the postnatal development of lower motor neurons during crucial periods of activity-dependent circuit modifications. We have generated Cre-dependent conditional Erk/MAPK mutant mice that exhibit either loss or gain of Erk/MAPK signaling specifically in ChAT:Cre expressing lower motor neurons. Importantly, we found that Erk/MAPK is necessary for the development of neuromuscular junction morphology by the second postnatal week. In contrast, we were unable to detect a significant difference in lower motor neuron development in Erk/MAPK gain-of-function mice. The data suggests that Erk/MAPK plays an important role in postnatal lower motor neuron development by regulating the morphological maturation of the neuromuscular junction. / Dissertation/Thesis / Masters Thesis Biology 2017

Les récentes évolutions en microélectronique nécessitent une attention particulière lors de la conception d’un circuit. Depuis les noeuds technologiques de quelques dizaines de nanomètres, les contraintes de consommation deviennent prépondérantes. Pour répondre à ce problème, les concepteurs se penchent aujourd’hui sur l’utilisation d’architectures multi-coeurs hétérogènes incluant des accélérateurs matériels dotés d’une grande efficacité énergétique. Le maintien des spécifications d’un circuit apparait également essentiel à l’heure où sa fabrication est de plus en plus sujette à la variabilité et aux défauts. Il existe donc un réel besoin pour des accélérateurs robustes. Les architectures neuromorphiques, et notamment les réseaux de neurones à impulsions, offrent une bonne tolérance aux défauts, de part leur parallélisme massif, et une aptitude à exécuter diverses applications à faible coût énergétique. La thèse défendue se présente sous deux aspects. Le premier consiste en la conception d’un neurone analogique robuste et à son intégration dans un accélérateur matériel neuro-inspiré à des fins calculatoires. Cet opérateur mathématique à basse consommation a été dimensionné puis dessiné en technologie 65 nm. Intégré au sein de deux circuits, il a pu être caractérisé dans l’un d’entre eux et ainsi démontrer la faisabilité d’opérations mathématiques élémentaires. Le second objectif est d’estimer, à plus long terme, l’impact des nouvelles technologies sur le développement de ce type d’architecture. Ainsi, les axes de recherches suivis ont permis d’étudier un passage vers un noeud technologique très avancé, les opportunités procurées par des Through-Silicon-Vias ou encore, l’utilisation de mémoires résistives à changement de phase ou à filament conducteur. / Due to the latest evolutions in microelectronic field, a special care has to be given to circuit designs. In aggressive technology nodes down to dozen of nanometres, a recent need of high energy efficiency has emerged. Consequently designers are currently exploring heterogeneous multi-cores architectures based on accelerators. Besides this problem, variability has also become a major issue. It is hard to maintain a specification without using an overhead in term of surface and/or power consumption. Therefore accelerators should be robust against fabrication defects. Neuromorphic architectures, especially spiking neural networks, address robustness and power issues by their massively parallel and hybrid computation scheme. As they are able to tackle a broad scope of applications, they are good candidates for next generation accelerators. This PhD thesis will present two main aspects. Our first and foremost objectives were to specify and design a robust analog neuron for computational purposes. It was designed and simulated in a 65 nm process. Used as a mathematical operator, the neuron was afterwards integrated in two versatile neuromorphic architectures. The first circuit has been characterized and performed some basic computational operators. The second part explores the impact of emerging devices in future neuromorphic architectures. The starting point was a study of the scalability of the neuron in advanced technology nodes ; this approach was then extended to several technologies such as Through-Silicon-Vias or resistive memories.

abstract: Gene manipulation techniques, such as RNA interference (RNAi), offer a powerful method for elucidating gene function and discovery of novel therapeutic targets in a high-throughput fashion. In addition, RNAi is rapidly being adopted for treatment of neurological disorders, such as Alzheimer's disease (AD), Parkinson's disease, etc. However, a major challenge in both of the aforementioned applications is the efficient delivery of siRNA molecules, plasmids or transcription factors to primary cells such as neurons. A majority of the current non-viral techniques, including chemical transfection, bulk electroporation and sonoporation fail to deliver with adequate efficiencies and the required spatial and temporal control. In this study, a novel optically transparent biochip is presented that can (a) transfect populations of primary and secondary cells in 2D culture (b) readily scale to realize high-throughput transfections using microscale electroporation and (c) transfect targeted cells in culture with spatial and temporal control. In this study, delivery of genetic payloads of different sizes and molecular characteristics, such as GFP plasmids and siRNA molecules, to precisely targeted locations in primary hippocampal and HeLa cell cultures is demonstrated. In addition to spatio-temporally controlled transfection, the biochip also allowed simultaneous assessment of a) electrical activity of neurons, b) specific proteins using fluorescent immunohistochemistry, and c) sub-cellular structures. Functional silencing of GAPDH in HeLa cells using siRNA demonstrated a 52% reduction in the GAPDH levels. In situ assessment of actin filaments post electroporation indicated a sustained disruption in actin filaments in electroporated cells for up to two hours. Assessment of neural spike activity pre- and post-electroporation indicated a varying response to electroporation. The microarray based nature of the biochip enables multiple independent experiments on the same culture, thereby decreasing culture-to-culture variability, increasing experimental throughput and allowing cell-cell interaction studies. Further development of this technology will provide a cost-effective platform for performing high-throughput genetic screens. / Dissertation/Thesis / Ph.D. Bioengineering 2012

As correntes subliminares determinam propriedades da membrana neuronal, tais como a resistência de entrada (Rin) e a constante de tempo (tm). Nesta tese, estudamos mecanismos pelos quais duas correntes subliminares (corrente ativada por hiperpolarização, Ih, e corrente de sódio persistente, INaP) determinam Rin e tm em dois tipos de neurônio: neurônio fusiforme do núcleo coclear dorsal e célula piramidal da região CA1 do hipocampo. A tese está dividida em três partes: a primeira estuda como a Ih atua concomitantemente com a corrente de potássio retificadora de entrada (IKIR) para manter Rin estacionária entre neurônios fusiformes com heterogeneidade de disparo (silenciosos, sem disparos espontâneos, e ativos, com disparos espontâneos regulares). Na segunda parte, usa-se uma combinação de modelagem computacional com a técnica experimental de dynamic-clamp em neurônios piramidais de fatias hipocampais para mostrar que a criação de uma região de inclinação negativa na curva I/V (condutância de inclinação negativa) pela ativação rápida da INaP é responsável pelo aumento de Rin e tm e pela amplificação e prolongamento dos potenciais pós-sinápticos das células. Finalmente, a terceira parte estabelece o mecanismo pelo qual a INaP e Ih controlam a tm da célula. Para isso, propomos um novo conceito denominado \"condutância de inclinação dinâmica\" que leva em consideração a cinética das correntes e explica os efeitos observados das cinéticas de Ih e INaP sobre tm. Com base nos resultados, prevemos que uma Ih com cinética rápida atenua e encurta os potenciais pós-sinápticos excitatórios muito mais que uma Ih com cinética lenta. / Subthreshold currents determine the neuronal membrane properties, such as the input resistance (Rin) and the membrane time constant (tm). In this thesis, we studied the mechanisms by which two subthreshold currents (the hyperpolarization-activated current, Ih, and the persistent sodium current, INaP) determine Rin and tm in two types of neurons: the fusiform neuron of the dorsal cochlear nucleus and the pyramidal cell of the CA1 region of the hippocampus. The thesis is divided in three parts: the first part studies how Ih acts concomitantly with the inwardly rectifying potassium current (IKIR) to equalize Rin among fusiform neurons with firing heterogeneity (quiet, without spontaneous firing and active, with regular spontaneous firing). In the second part, we used a combination of computational modeling with the experimental technique dynamic-clamp in pyramidal cells of hippocampal slices to show that the creation of a negative slope region in the I/V curve (negative slope conductance) by the fast activation of the INaP is responsible for the increase of Rin and tm, and for the amplification and prolongation of postsynaptic potentials in these cells. Finally, the third part establishes the mechanism whereby INaP and Ih control tm in the cell. For this, we propose a new concept called \"dynamic slope conductance\", which takes into consideration the current kinetics and explains the observed effects of Ih and INaP kinetics on tm. Based on the results, we predict that an Ih current with fast kinetics attenuates and shortens excitatory postsynaptic potentials strongly than an Ih current with slower kinetics.