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No description available.

Programmed cell death (PCD) is a highly evolved mechanism of cellular suicide that is aberrantly activated following neural injury. Two fundamental PCD signaling pathways termed the extrinsic (caspase-8-mediated) and intrinsic (caspase-9-mediated) pathways, have been described. While each pathway is initiated by distinct cellular stimuli, both pathways culminate in the activation of downstream executioner caspases. Previous efforts to isolate the in vivo contribution of each pathway have been hindered by the embryonic lethality of casp8 and casp9 null mice. In the present study, I overcame this obstacle to directly assess the contribution of each pathway following two well-characterized forms of acute neural injury; excitotoxic destruction of CA1 pyramidal neurons, and the loss of motor neurons following facial nerve transection. To determine the role of caspase-8, I constructed several lines of mice in which caspase-8 was conditionally ablated within the relevant neuronal populations. The results obtained from these animals definitively demonstrate that caspase-8 is not required by either motor neurons or CA1 pyramidal neurons to undergo PCD following injury. Therefore, these findings have provided the first direct experimental evidence to counter the widely held dogma of caspase-8 as the central effector of death receptor-mediated signaling within neurons. With respect to the intrinsic pathway, several lines of evidence suggest that the apoptosome predominantly regulates the death of motor neurons. I tested this hypothesis by performing facial axotomies in mice containing a point mutation introduced (“knocked in”) into the genomic locus of cytochrome c which abolishes its ability to activate the intrinsic pathway. Homozygous cytochrome c knock-in mice displayed a significant enhancement in motor neuron survival in comparison to control littermates following injury. However, the level of motor neuron protection differed from that previously reported in mice either overexpressing anti-apoptotic or lacking pro-apoptotic members of the Bcl-2 family. Therefore, the results of this study directly demonstrate the influence of the apoptosome on injury-induced neuronal PCD isolated from upstream Bcl-2 family-mediated effects. In addition, my results have provided the first evidence that activation of the apoptosome is required for the release of apoptosis inducing factor (AIF) from the mitochondria of injured motor neurons in vivo.

No description available.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are defined by infectivity and by the pathological damage they produce in the central nervous system (CNS), typically involving spongiform degeneration or vacuolation, deposition of abnormal PrP (PrPSc), glial activation and neuronal loss. Much of our understanding of the TSEs has derived from the study of murine scrapie models. The molecular basis of pathological changes is not clear, in particular the relationship between the deposition of PrPSc and neuronal dysfunction. A typical feature of TSE disease is neuronal loss, although the mechanisms leading to this loss are poorly understood. Apoptosis has been proposed as an important mechanism of TSE associated cell death, but which pathways are involved are still to be determined. The main aims of this thesis are to investigate the progression of the characteristic neuropathological changes observed in the TSE infected brain and to analyse the mechanisms involved in neuronal loss. In this study two contrasting scrapie mouse models were used : the ME7/CV model , and the 87V/VM model in which neuronal loss is targeted to different areas of the hippocampus, the CA1, and CA2 respectively. The role of the caspase-dependent pathway of apoptosis in the neuronal loss was investigated. The results of analysis of pro-apoptotic markers of disease in the two scrapie mouse models differed. The results observed in the ME7/CV scrapie mouse model suggest that apoptosis may not be the main mechanism of neuronal loss, whereas the 87V/VM model showed some indication that apoptosis may be involved. Detailed studies in the progression of neurodegenerative changes in the ME7/CV scrapie mouse model revealed that the initial pathological change observed in the hippocampus was the deposition of PrPSc followed by a glial response, spongiform change and subsequent neuronal degeneration. The role of the cytoskeleton and synaptic dysfunction in the neuronal damage observed in the CA1 of the ME7 infected hippocampus was analysed. Cytoskeletal disruption was observed in the post-synaptic dendritic spine, and the apical dendrites of CA1 neurons at 160days, a time point at which neurons are known to be lost. Changes in the expression of the pre-synaptic protein, synaptophysin and the post-synaptic protein PSD-95 were not observed until the terminal stage of disease when the neuronal loss is profound. In conclusion, this research suggests that the mechanisms of neuronal loss may follow different biochemical pathways, which might not necessarily involve an apoptotic mechanism. Cytoskeletal disruption in the post-synaptic dendritic spine plays a major role in the neuronal dysfunction observed in ME7 infected CA1 neurons, although the post synaptic density does not seem to be involved .Pre-synaptic changes and disruption to the innervation of CA1 neurons is not apparent until the end stages of disease. The trigger for this cytoskeletal disruption and the subsequent neuronal loss may be the early deposition of PrPSc in the extracellular space but the precise mechanisms involved are still to be elucidated. The identification of the key events involved in the mechanisms of neruodegeneration in TSE diseases may lead to the development of therapeutic strategies to inhibit the neurodegenerative process.

En dehors de l'hypothèse de la cascade amyloïde qui domine l'activité de recherche liée à la maladie d'Alzheimer, d'autres hypothèses ont été proposées pour expliquer la pathogénie de la maladie. Parmi celles-ci, le stress oxydant, le dysfonctionnement mitochondrial ou encore l'altération cholinergique ont été suggérés car ils sont à la base des mécanismes qui apparaissent précocement et contribuent de façon importante à la physiopathologie de la maladie. Notre projet propose d'étudier ces facteurs à travers une évaluation phénotypique histopathologique et neurochimique dans deux mutations du gène PS-1 (souris PS1/A246E et PS1/I213T) caractérisées par une surexpression du peptide Aß-42 sans amyloïdose et des troubles comportementaux peu sévères mimant des stades précoces de la maladie. Une évaluation des bases d'ADN oxydées a permis la mise en évidence d'un dommage oxydatif précoce dans des régions variées pour lesquelles le taux de peptide Aß est généralement augmenté. Des cartographies cérébrales des activités de la cytochrome oxydase et de l'acétylcholinestérase ont servi respectivement à l'étude de la fonction métabolique neuronale et de l'innervation cholinergique. Les modifications d'activité métabolique obtenues ne signent pas d'altération intrinsèque de la cytochrome oxydase mais témoignent de phénomènes compensatoires indépendants du dommage oxydatif tissulaire. Les perturbations cholinergiques sont importantes dans les deux mutations impliquant les circuits d'activation de l'attention et de la mémorisation. / In addition to the amyloid cascade hypothesis, predominant in Alzheimer's disease research, other hypotheses have been proposed to explain its pathogenesis. Among those, oxidative stress, mitochondrial dysfunction, and cholinergic alterations were suggested, based on early onset signs contributing in an important way to the physiopathology. Our project studied these factors via phenotypic evaluation in the context of histopathology and neurochemistry in two PS-1 mutations: PS1/A246E transgenic and PS1/I213T knock-in mice, characterized by overexpressed Aß-42 peptide without amyloidosis, together with mild behavioral impairments mimicking early onset Alzheimer's disease. Both mutants showed oxidative damage, as measured by DNA oxidation in regions with increased Aß. Brain regional cartographies of cytochrome oxidase and acetylcholinesterase reflected respectively neuronal metabolism and cholinergic innervation. There was no intrinsic modulation of cytochrome oxidase but rather compensatory phenomena independent of oxidative damage. Cholinergic alterations were important in each mutation, particularly in circuits associated with selective attention and memory.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system, playing critical roles in basal synaptic transmission and the molecular correlates of learning and memory, long-term potentiation and long-term depression. However, glutamate is also neurotoxic during prolonged exposure and the dysfunction of the glutamatergic system has been implicated in most neurological disorders, including stroke and epilepsy, and in certain neurodegenerative diseases, including Alzheimer’s disease. In these conditions, an increased concentration of extracellular glutamate causes an over-activation of local ionotropic glutamate receptors that trigger neuronal cell death (excitotoxicity). In this study, we have used dissociated hippocampal neurons cultured on coverslips and within novel microfluidic devices to study neuronal responses, both functional and morphological, to prolonged exposure to glutamate. We find that high glutamate concentrations evoke a rapid retraction of dendritic spines, the collapse of microtubules, the formation of dendritic beads and the inhibition of basal neurotransmitter release. These responses have been identified in many neurological disorders where excitotoxicity is reported, suggesting they may be a sign of imminent cell death. However, the development of dendritic beads and the inhibition of network activity also occurs at subtoxic concentrations of glutamate and neuronal morphological changes recover rapidly post-insult. We therefore hypothesised that beading and the inhibition of neurotransmitter release may be a protective mechanism and render neurons resistant to subsequent glutamatergic insults. However, a subtoxic stimulation is not protective against a subsequent excitotoxic insult delivered immediately afterwards. However, given that neurotransmitter release can confer protection to neurons, it is possible that protection is realised, not on the neurons exposed to the subtoxic insult, but on those neurons with which they communicate, as a ‘warning’ signal. To assess the impact of a localised insult to a wider neuronal network, hippocampal neurons were cultured in novel microfluidic devices, to environmentally isolate neuronal populations, whilst preserving synaptic contacts between them. We observe that bystander naïve neurons downstream of a localised excitotoxic insult succumb to a secondary, activity-dependent, spreading toxicity. In addition, we reveal a novel mechanism by which neuronal networks also transmit a rapid and robust (albeit transient) protection from excitotoxicity. The protective phenotype acquired by neurons during this protective process requires neuronal inhibitory activity to quench overexcitation, along with the retraction of dendritic spines and/or dendritic beading. Therefore, we highlight a dichotomous role that dendritic beading plays following a direct glutamatergic insult (large beads) and as a result of GABAergic recruitment in downstream neurons (small beads). We determine that a network neuroprotective capacity exists that limits spreading toxicity, which may be recruited from a distal site even after an excitotoxic insult has occurred. Together, we may have identified a new therapeutic opportunity to limit on-going brain damage in conditions of acute neuronal injury.

Major Depressive Disorder (MDD) is exceedingly prevalent and considered to be one of the leading cause of disability worldwide. Depression is also a heterogeneous disorder characterized by complex diagnotic approaches with a lack of diagnostic biomarker, an inconsistent response to treatment, no established mechanism, and affecting multiple physiological systems such as endocrine, immunological and cardiovasular as well. The growing impact of the analysis of complex signals on biology and medicine is fundamentally changing our view of living organisms, physiological systems, and disease processes. In this endeavour, the basic challenge is to reveal how the coordinated, dynamical behavior of cells and tissues at the macroscopic level, emerges from the vast number of random molecular interactions at the microscopic level. In this way, the fundamental questions could be: (i) how physiological systems function as a whole, (ii) how they transduce and process dynamical information, (iii) how they respond to external stimuli, and mostly (iv), how they change during a pathological processus. These challenges are of interest from a number of perspectives including basic modeling of physiology and practical bedside approaches to medical and risk stratification. The general purpose of this thesis, therefore, is to study physiological time series to provide a new understanding of sleep dynamics in health, specifically as they apply to the pathological condition of MDD. More precisely: (1) to quantitatively characterize the complex, nonlinear behaviour of cardiovascular (ECG) and electroencephalographic (EEG) time series during sleep, in health and in MDD. This project will test the hypotheses that both the sleep EEG and ECG detects reorganization in the system dynamics in patient suffering from depression. (2) To develop new diagnostic and prognostic tests for MDD, by detecting and extracting “hidden information” in the ECG and EEG datasets. Three different methods are introduced in this thesis for the analysis of dynamical systems. The first one, detrended fluctuation analysis, can reveal the presence of long-term correlations ("memory" in the physiological system) even when embedded in non-stationary time series. Graph theoretical measures were then applied to test whether disrupting an optimal pattern ["small-world network"] of functional brain connectivity underlies depression. Finally, multiscale entropy method, which is aimed at quantifying the complexity of the systems' output resulting from the presence of irregular structures on multiple scales, was applied on the ECG signal. The results indicate that healthy physiologic systems, measured through the EEG and the ECG signals, are the most complex. According to the decomplexification theory, the depressive disease model exhibits a loss of system complexity, with potential important applications in the development and testing of basic physiologic models, of new diagnostic and prognostic tools in psychiatry, and of clinical risk stratification.

The study of cultured neuronal networks has opened up avenues for understanding the ion channels, receptor molecules, and synaptic plasticity that may form the basis of learning and memory. The hippocampal neurons prepared from Wistar rats and put in culture, show, after a few days, spontaneous activity with typical electrophysiological pattern ranging from stochastic spiking to synchronized bursting. Using a multi-electrode array (MEA) having 64 electrodes, the electrophysiological signals are acquired, and connectivity maps are constructed using correlation matrix to understand how the neurons in a network communicate during the burst. The response of the neuronal system to epilepsy caused by induced glutamate injury and subsequent exposure of the system to phenobarbital to form different connectivity networks is analyzed in this study. The correlation matrix of the neuronal network before and after administering glutamate as well as after administering phenobarbital is used to understand the neuronal and network level changes that take place in the system. In order to interface a neuronal network to a physical world, the major computations to be performed are noise removal, pattern recovery, pattern matching and clustering. These computations are to be performed in real time. The system should be able to identify a pattern and relate a physical task to the pattern in about 200-400 ms. Algorithms have been developed for the implementation of a real-time neuronal system on a multi-node digital processor system.

The structure and functions of brain show sexual dimorphism in vertebrates. Brain sexual differentiation is resulted from the neural development. The neural development is determined not only by the genetic regulation, but also by the extrinsic environmental influences. Serotonin (5-hydroxytryptamine, 5-HT ) functions as a neurotransmitter or/and neuromodulator in the central nervous system. Serotonin plays a role in the neural development via serotonin receptors. The sexual differentiation of tilapia is influenced by water temperature. The lower temperature induces a higher proportion of female while the elevated temperature induces a higher proportion of male in tilapia. In the present study, the influence of temperature on the proliferation of the neurons was investigated. These results show that the proliferation of neurons are varied with the temperature. The elevated temperature influences the proliferation of neurons via central serotonin system. Serotonin 1A receptor is involved in the serotonin-induced proliferation of neuron.