Amyotrophic Lateral Sclerosis is the most common type of motor neuron disease, characterized by progressive degeneration of the upper and lower motor neurons. Sufferers present with symptoms of muscle weakness and this quickly develops on to paralysis and finally death due to respiratory failure within 5 years of disease onset. Although the majority of cases are sporadic, about 10% are familial and it is hoped that through the investigation of these few cases a greater understanding of the disease process, the reasons for its delayed onset and vulnerability of motor neurons will be achieved. Recently a novel mutation linked to ALS was discovered in an evolutionary conserved protein named Vesicle associated membrane protein (VAMP) associated protein B (VAPB). VAPB is an integral type II membrane protein localised at the Endoplasmic Reticulum and thought to have a role in protein transport. The orthologue in Drosophila has been shown to be involved in the homeostatic regulation of bouton formation at the Neuromuscular Junction through an association with the microtubule network. To elucidate the mechanism through which this protein causes ALS, Pennetta et al have created a Drosophila model of the disease by expressing the mutated orthologue in the fly. To complement this model, I have undertaken a number of biochemical experiments to look for potential interactors of the VAP proteins. The yeast two hybrid system utilises the yeast GAL4 transcriptional activator to indicate a protein interaction within a yeast cell and can be used to test a cDNA library for interactors. Through this technique a number of interesting binding partners have been found that may play crucial roles in the progression of the disease.

"Stable multi-day recordings from chronically implanted microelectrodes within the dorsolateral prefrontal cortex of two monkeys performing three Go/NoGo visual discrimination tasks (one requiring well-learned responses, two requiring learning) demonstrated that the majority of prefrontal neurons were 'functionally stable'. Action potentials of 94 neurons were stable over 2-9 days; 66/94 (70%) of these cells responded each day, 22/94 (23%) never responded significantly, and 6/94 (6%) responded one day but not the next. Of 66 responsive neurons, 55 were selective for either Go or NoGo trials, individual stimuli, or eye movements." (Greenberg and Wilson, 2004) Selectivity was maintained, for 46/55 neurons across all recording days. Response strength (baseline vs. post-stimulation firing rates) and event-related response timing also displayed stability. Stability generalized across neuronal response type suggesting that functional stability is a general property. Long-term recordings from other studies supported similar conclusions suggesting that neurons throughout the brain are functionally stable. Single-day recordings from different neurons within the same cortical regions demonstrated neuronal response flexibility while monkeys learned associations among visual cues, and Go/NoGo behavioral responses. Of 116 neurons, 57 (49%) displayed significant change points in firing rates during novel learning (n=18), reversal learning (n=12), or both tasks (n=27). Six of 57(10.5%) neurons had firing rates changes prior to learning and might have been causally related to the monkeys' behavioral changes. However, only 18/152 (12%) of the total number of firing rate changes occurred prior to the monkeys' learning meaning that most appeared to be the consequences of learning rather than the causes.

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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.

The goal of this study was to identify critical regions of a novel gene, Nance-Horan syndrome-like 1b (nhsl1b). It was previously discovered that C-terminal truncation of the Nhsl1b protein in nhsl1b mutants resulted in a loss of migration in the facial motor neurons of the hindbrain (Walsh et al. 2011). As nhsl1b expresses many isoforms, multiple targets were investigated in order to determine which transcript bears the largest impact on the motor neurons. Using confocal microscopy to observe immunostained embryos, we examined a mutation in an nhsl1b transcript that encodes a WHD, a domain that is known to function within the actin nucleation and polymerization pathways. In situ hybridization and injection of antisense morpholino oligonucleotides indicate that it is not the WHD but another transcript (ex1bnhsl1b) that is necessary for migration. The control experiments for rescuing the mutant phenotype have successfully been performed, but inducing expression of full length nhsl1b in the nhsl1b mutants is proving difficult.

The time accuracy of the auditory neuronal pathway in its sound localization branch is high, compared to other sensory systems. The time differences in the sound arrival between the left and right ear are distinguished by the neural circuit in this branch. The accuracy achieved here is in the order of tens of microseconds. This phenomenon has not yet been definitively clarified. In this master thesis, a model of a neuron central to this neural circuit is presented. This neuron is called binaural (neuron of the two ears) and is located in the medial superior olive (MSO) neural nucleus. The properties of the MSO neuron are described. Specifically, the neuron acts as a coincidence detector, and this is necessary for the circuit functioning. Main result of the thesis is the theory explaining how the function of the coincidence detector can be described based on the interaction of the post-synaptic potentials on the spike-response model neuron. Generality and implications for the auditory pathway are then discussed.

Les micro-ARNs (miRNAs) sont de petits ARNs (20-25 nt) qui ont un rôle important dans les mécanismes d'interférence ARN. Les miRNAs sont des inhibiteurs de l'expression génique qui interviennent au niveau post-traductionnel en s'hybridant à des sites spécifiques de leurs ARNm cibles. Ce mécanisme induit la dégradation de l'ARNm ou l'inhibition de sa traduction. Puisque l'hybridation partielle du miRNA est suffisante pour induire une inhibition, chaque miRNA peut avoir des centaines de cibles. Les miRNAs sont impliqués dans de nombreuses fonctions biologiques et en particulier dans processus neuronaux. Plus de la moitié des miRNAs connus sont exprimés dans le cerveau de mammifère avec une distribution spécifique du miRNA considéré. A l'échelle sub-cellulaire il y a également une distribution hétérogène des miRNAs. De plus, il a été montré récemment une implication des miRNAs dans la régulation de la traduction locale dans les neurones. En effet, des miRNAs et des protyeines impliquées dans la biogenèse et la fonction des miRNAs ont été retrouvés dans le soma, les dendrites et les axones. Il a été montré que la dérégulation des miRNAs été impliquée dans de nombreux mécanismes pathologiques. Cette thèse a pour objectif de révéler le rôle des miRNAs dans la plasticité synaptique. Nous avons étudié l'implication des miRNAs dans les mécanismes de la plasticité synaptique homéostatique et dans la plasticité dysfonctionnelle rencontrée en condition de douleur cancéreuse.Notre hypothèse était que la régulation de la traduction locale des récepteurs AMPA dans les dendrites en condition d'homéostasie synaptique implique les miRNAs. Par bio-informatique, qRT-PCR et test luciférase, nous avons identifié le miRNA miR-92a comme régulateur de la traduction de l'ARNm de GluA1. Des immunomarquages des récepteurs AMPA et des enregistrements des courants miniatures AMPA montrent que miR-92a régule spécifiquement l'incorporation synaptique de nouveau récepteurs AMPA contenant GluA1 en réponse à un blocage de l'activité synaptique. La douleur est un symptôme très fréquemment associé au cancer et constitue un challenge pour les médecins puisque aucun traitement spécifique et efficace n'existe. C'est sans doute le résultat d'un manque de connaissances des mécanismes moléculaires responsables de la douleur cancéreuse. En combinant les screening des miRNA et des ARNm, nous avons mis en évidence une voie de régulation impliquant miR-124, un miRNA enrichi dans le système nerveux. Ainsi, dans un modèle de douleur cancéreuse chez la souris, la diminution de miR-124 est associée à une augmentation de ces cibles : calpain 1, synaptopodine et tropomyosine 4. Toutes ces protéines ont précédemment été identifiées comme des molécules clef de la fonction et de la plasticité synaptique. Des experiences in vitro ont confirmé que miR-124 exercait une inhibition multiple de calpain 1, synaptopodine et tropomyosine 4. La pertinence clinique de cette découverte a été vérifiée par le screening du liquide cérébro-spinal de patients souffrant de douleur cancéreuse qui montre également une diminution de miR-124. Ce résultat suggère un fort potentiel thérapeutique du ciblage de miR-124 dans les douleurs cancéreuses. Enfin, l'injection intrathécale de miR-124 dans des souris cancéreuses a permis de normaliser l'expression de la synaptopodine et de stopper la douleur cancéreuse lors de la phase initiale de la maladie. / MicroRNAs (miRNAs) are a type of small RNA molecules (21-25nt), with a central role in RNA silencing and interference. MiRNAs function as negative regulators of gene expression at the post-transcriptional level, by binding to specific sites on their targeted mRNAs. A process results in mRNA degradation or repression of productive translation. Because partial binding to target mRNA is enough to induce silencing, each miRNA has up to hundreds of targets. miRNAs have been shown to be involved in most, if not all, fundamental biological processes. Some of the most interesting examples of miRNA activity regulation are coming from neurons. Almost 50% of all identified miRNAs are expressed in the mammalian brain. Furthermore, miRNAs appear to be differentially distributed in distinct brain regions and neuron types. Importantly, miRNAs are reported to be differentially distributed at the sub-cellular level. Recently, miRNAs have been suggested to be involved in the local translation of neuronal compartments. This has been derived from the observations reporting the presence of miRNAs and the protein complexes involved in miRNA biogenesis and function in neuronal soma, dendrites, and axons. Deregulation of miRNAs has been shown to be implicated in pathological conditions. The present thesis aimed at deciphering the role of miRNA regulation in neuronal plasticity. Here we investigated the involvement of miRNA in synaptic plasticity, specifically in homeostatic synaptic plasticity mode. In addition, we investigated the involvement of miRNAs in the maladaptive nervous system state, specifically, in bone cancer pain condition.We hypothesized that local regulation of AMPA receptor translation in dendrites upon homeostatic synaptic scaling may involve miRNAs. Using bioinformatics, qRT-PCR and luciferase reporter assays, we identified several brain-specific miRNAs including miR-92a, targeting the 3’UTR of GluA1 mRNA. Immunostaining of AMPA receptors and recordings of miniature AMPA currents in primary neurons showed that miR-92a selectively regulates the synaptic incorporation of new GluA1-containing AMPA receptors during activity blockade.Pain is a very common symptom associated with cancer and is still a challenge for clinicians due to the lack of specific and effective treatments. This reflects the crucial lack of knowledge regarding the molecular mechanisms responsible for cancer-related pain. Combining miRNA and mRNA screenings we were able to identify a regulatory pathway involving the nervous system-enriched miRNA, miR-124. Thus, miR-124 downregulation was associated with an upregulation of its predicted targets, Calpain 1, Synaptopodin and Tropomyosin 4 in a cancer-pain model in mice. All these targets have been previously identified as key proteins for the synapse function and plasticity. Clinical pertinence of this finding was assessed by the screening of cerebrospinal fluid from cancer patient suffering from pain who presented also a downregulation of miR-124, strongly suggesting miR-124 as a therapeutic target. In vitro experiments confirmed that miR-124 exerts a multi-target inhibition on Calpain 1, Synaptopodin and Tropomyosin 4. In addition, intrathecal injection of miR-124 was able to normalize the Synaptopodin expression and to alleviate the initial phase of cancer pain in mice.

L'excitabilité est une propriété bien connue des neurones biologiques. Il s'agit d'une réponse de type tout-ou-rien à une perturbation au delà d'un seuil caractéristique appelé seuil excitable. D'autres propriétés importantes existent dans les neurones comme les périodes réfractaires et la sommation temporelle ou spatiale de stimuli d'entrée.L'excitabilité a été étudiée dans certains composants actifs à semiconducteur et notamment les composants à semiconducteurs III-V. Leurs propriétés neuro-mimétiques pourraient permettre de traiter l'information de façon tout-optique avec une grande bande passante et une faible consommation.Grâce aux nouvelles techniques de micro-nano fabrication, il est devenu possible de fabriquer des micropiliers lasers à absorbant saturable. Ces micropiliers pourraient permettre la réalisation de réseaux de micropiliers couplés excitables analogues à des réseaux de neurones photoniques.Dans cette thèse j'ai étudié les propriétés neuro-mimétiques de micropiliers lasers à absorbant saturable intégré. Les principaux résultats de cette thèse sont les suivants : 1) la technique de fabrication des micropiliers a été améliorée conduisant à une augmentation de leur durée de vie et une diminution du seuil laser. 2) des propriétés de base des neurones biologiques, comme l'excitabilité, l'existence des périodes réfractaires, la sommation temporelle, ont été mises en évidence expérimentalement et analysées à l'aide du modèle de Yamada. 3) des effets de propagation d'excitations ont été démontrés dans des structures unidimensionnelles : des lasers ligne et des chaînes de micropiliers couplés.La démonstration des propriétés neuromimétiques de micropiliers lasers à absorbant saturable et la mise en évidence de la propagation d'excitations ouvrent la voie à la réalisation de réseaux de micropiliers couplés pour les traitements neuromimétiques des signaux qui pourront être exploités pour de la logique codée à l'aide de pics excitables ainsi que pour du stockage d'information dans des circuits mémoires tout-optiques. / Excitability is a well known property of biological neurons. In excitable systems, the response to a perturbation above the excitable threshold is of all-or-none type. Other properties exist in neurons such as the refractory periods and temporal or spatial summation of input stimuli.Excitability has been demonstrated in many III-V semiconductor material devices. Thanks to their nonlinear properties it could be possible to realize neuromimetic and all-optical signal processing with high speed and low energy consumption. Thanks to progress in fabrication techniques it is possible to fabricate high quality micropillar laser with saturable absorber. Thus, using micropillars it could be possible to realize neural photonic networks analog to neural networks.In this thesis work, I studied neuron-like properties of a micropillar laser with a saturable absorber. My main results are : 1) fabrication of micropillars has been improved leading to an increase of their robustness and a reduction of the laser threshold. 2) well known properties of biological neurons, such as excitability, existence of refractory periods, temporal summation, have been demonstrated experimentally and have been numerically analyzed with the Yamada model. 3) propagation effects of excitations have been demonstrated in one-dimensional structures : wire lasers and chains of coupled micropillars.The demonstration of neuromimetic properties in micropillar lasers with saturable absorber and the evidence of propagation of excitations pave the way to neuromorphic networks based on coupled micropillars for neuromimetic signal processing like information encoding with excitable pulses and realization of optical memories.

The anterior cingulate cortex (ACC) can influence emotional and motivational states in primates by its dense connections with many neocortical and subcortical regions. Pyramidal neurons serve as the basic building blocks of these neocortical circuits, which have been extensively studied in other brain regions, but their morphological and electrophysiological properties in the primate ACC are not well understood. In this study, we used whole-cell patch clamp and high-resolution laser scanning confocal microscopy to reveal the general electrophysiological properties and detailed morphological features of layer 2 and 3 pyramidal neurons in ACC (area 24/32) of the rhesus monkey. Neurons from both layers had similar passive membrane properties and action potential properties. Morphologically, dendrites of layer 3 ACC neurons were more complex than those of layer 2 neurons, by having dendrites with longer total dendritic lengths, more branch points and dendritic segments, spanning larger convex hull volumes. This difference in total dendritic morphology was mainly due to the apical dendrites. In contrast, the basal dendrites displayed mostly similar features between the two groups of neurons. However, while apical dendrites extend to the same layer (layer 1), the basal dendrites of layer 3 extended into deeper layers than layer 2 because of the difference in soma-pia distance. Thus, basal dendrites of the two groups of neurons receive different laminar inputs. Analysis of spines showed that more spines were found in neurons of layer 3 apical dendritic arbors than layer 2 neurons. However, the apical spine densities were similar between neurons in the two layers. Thus, while higher spine number suggests that layer 3 neurons receive more excitatory input than layer 2 neurons, the similar spine density suggests similar spatial and temporal summation of these inputs. The combined effects of increased number of excitatory input and higher dendritic complexity in layer 3 than in layer 2 ACC neurons suggest the additional information received by layer 3 neurons, especially in the apical dendrites, might undergo more complex integration.