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Increasing the physiological relevance of cortical culture experiments via embodiment and manipulation of endogenous cholinergic toneHammond, mark W. January 2012 (has links)
Cortical cultures are increasingly implemented as an in vitro model of cortical networks for the study of network level learning and memory using planar multi-electrode arrays. However the recurrent, culture-wide synchronous activity (termed 'global bursts') exhibited by cortical cultures is not representative of the predominantly asynchronous activity characteristic of the intact cortex. Moreover, it has been demonstrated that such activity can interfere with the induction of plasticity and thus result in findings which cannot be related to networks in vivo. Critically, the mechanisms underlying such non-physiological global burst activity have been proposed to arise from incomplete development of the inhibitory network, as a result of the absence of afferent input during synaptic development. Therefore, the hypothesis that that re-introduction of afferent activity will, at least partially, rectify such developmental defects in the synaptic development is proposed. A recording environment was constructed to allow long term embodiment of a cortical culture using actuators and sensors of a mobile robot. Subsequently, the influence of electrical stimulation throughout synaptic development was investigated and found to alter spontaneous activity from global burst activity to a predominantly asynchronous state more comparable to that in vivo. Finally, the presence of significant endogenous, age dependent, cholinergic tone was demonstrated in these cultures and found to contain both muscarinic and nicotinic components. The presence of cholinergic tone and the striking comparability of its modulatory affect to that in vivo, increases the physiological relevance of the cortical culture platform, both within and outside of Animat paradigms. Moreover, such tone also provides a subtle and dynamic method by which plasticity and information flow could be experimentally modulated, in addition to overall excitability, in studies of mechanisms underlying learning and memory.
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Redox regulation through S-thiolation and S-nitrosylation of the BCAT proteins with insights in to the role these systems play in neuronal cellsColes, Steven John January 2008 (has links)
The human mitochondrial and cytosolic branched chain aminotransferases (hBCATm and hBCATc, respectively) are key metabolic enzymes that catalyse the reversible transamination of the nutritionally essential branched-chain amino acids. Both isozymes have a conserved redox active CXXC motif, unique among the mammalian aminotransferases. In the present study the effect of ^S-nitrosylation, iS-thiolation or disulphide bond formation on the functionality of the BCAT proteins was investigated.
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Synchronization and decoding in spiking neuronal networksRossoni, Enrico January 2007 (has links)
No description available.
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Ultrastructural localisation of the α4 nicotinic acetylcholine receptor subunit in rat nigro-striatal neuronsBradly, Amy Louise January 2003 (has links)
No description available.
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Manipulating gene expression in DRG sensory neuronesSeereeram, Anjan January 2005 (has links)
Mammalian sensory neurones that respond to tissue damaging stimuli are known as nociceptors. Activation of these neurones can induce a sensation of pain. The aim of this thesis was to develop methods to manipulate gene expression in rodent sensory neurones, in order to understand more about the molecular mechanisms involved in pain pathways. Three approaches were compared: viral gene delivery, antisense gene knock-down, and transgenic knock-out mice. Firstly, novel recombinant Herpes simplex viruses were used to infect sensory ganglia, and the efficiency of gene transfer assessed using histochemical markers. Secondly, antisense oligonucleotides directed against genes involved in determining neuronal excitability were delivered intrathecally, and the effects on gene expression and behaviour were assessed. Finally, two transgenic mouse lines in which the bacterial recombinase Cre was expressed downstream of sensory neuron specific promoters were analysed. These mice can be used to delete genes flanked by lox-p sites in a tissue-specific manner. The pattern of expression of Cre recombinase was assessed using reporter mice that express beta-galactosidase down stream of lox-p flanked stop (poly-adenylation) sites. Experiments with modified Herpes simplex viruses expressing green fluorescent protein (GFP) showed that injection of virus into the sciatic nerve resulted in more infected DRG neurones than footpad injection. A maximum transduction level of 7.9 1.3 % of all neurones was achieved one month after sciatic delivery. The majority of transduced neurones (67 1%) were large-diameter neurones. This subpopulation is not generally responsible for the transmission of noxious stimuli. Only 3.4 0.8% of peripherin positive neurones (a population that includes the majority of nociceptors) were successfully transduced. This level of transduction was too low to be used effectively. Vectors expressing VP22 a protein that enhances cell-to-cell viral spread were made, but were unstable. The use of these HSV vectors is thus limited to experimental situations where infection of only a few cells is adequate (for example, examining the function of secreted molecules). Experiments with labelled antisense probes gave much higher rates of neuronal transduction in comparison to viral transduction methods ( 80%). Unlike HSV-vectors however, there was no preferential transduction of neuronal cells, and all cell types were susceptible to oligonucleotide uptake. Intrathecal administration of antisense oligonucleotides directed against the annexin light chain protein p11 resulted in lowered levels of expression of the sodium channel Nav1.8 that requires p11 for insertion into the plasma membrane. Behavioural experiments were carried out to investigate the changes in both normal sensory function, and the alterations in inflammatory-related hyperalgesia after the administration of p11 antisense molecules. It was shown that this approach could successfully modulate the subcellular location of Nav1.8 protein. The results point to a dramatic and specific role for BDNF in the processing of noxious stimuli. In summary, it is clear that a variety of methods can be used to manipulate gene expression in sensory neurones to examine mechanisms involved in nociception. In this thesis, successful experiments using antisense oligonucleotides and nociceptor-specific Cre-expressing mice were carried out. It is concluded that the most effective route to manipulating nociceptor gene expression is the use of transgenic mice, but that antisense strategies have some advantages in terms of simplicity and speed. The use of HSV vectors is limited at present due to the low nociceptor transduction efficiency that can be achieved by relatively non-invasive delivery methods necessitating further improvements in vector design before their full potential can be realised.
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Neurotransmitter signalling to oligodendrocytesKáradóttir, Raghildur Póra January 2006 (has links)
Neurotransmitter signalling to neurons and glial cells plays a key role in brain development, information processing and pathological processes. This thesis focuses on neurotransmitter signalling to oligodendrocytes, the glial cells which provide myelin to speed the propagation of action potentials along neuronal axons. In cerebellar and corpus callosal slices, I used patch-clamping and immunocytochemistry to examine the properties of precursor, immature and mature oligodendrocytes, characterizing their morphology and basic electrical properties, their response to glutamate, GABA and other neurotransmitters, and the neurotransmitter receptor subunits that they express. In contrast to the currently held view, I found that oligodendrocytes express NMDA receptors. These receptors show extremely weak magnesium-block, allowing them to be activated at the resting potential, and they may be composed of NR1, NR2C and NR3 subunits. To investigate the role of these NMDA receptors in pathology, experiments on hippocampal neurons were first used to establish how best to block glycolytic and mitochondrial production of ATP to mimic the energy deprivation which occurs in ischaemia. Ischaemia-evoked glutamate release was found to activate oligodendrocyte NMDA and non-NMDA receptors. Although the normal role of the oligodendrocyte NMDA receptors may be to regulate myelination, they probably contribute to the glutamate-mediated damage which occurs to oligodendrocytes in periventricular leukomalacia (leading to cerebral palsy), stroke, spinal cord injury and multiple sclerosis. Block of these receptors may therefore offer a potential therapeutic approach to treating these disorders.
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Molecular dissection of ionotropic glutamate receptor delta-family interactions with trans-synaptic proteinsClay, Jordan Elliott January 2013 (has links)
Correct functioning of the brain relies upon the precise connectivity between the billions of neurons that make up this crucial organ. Aberrations in the formation of these elaborate neural networks lead to neurodegenerative and neuropsychiatric disorders. A synapse-spanning molecular triad, involving members of the Neurexin, Cbln and ionotropic glutamate receptor delta families of proteins, is crucial for the accurate formation and proper function of synapses in the cerebellum. This trans-synaptic complex has been implicated in the molecular mechanisms behind motor control and motor learning, and furthermore individual members have been linked to diseases such as Alzheimer’s, autism spectrum disorders and schizophrenia. The major findings presented in this thesis include: crystal structures of the amino-terminal domains (ATD) of the two members of the ionotropic glutamate receptor delta (iGluR-Delta) family, functional characterisation of the effects of disrupting the ATD interface in one member of the iGluR-Delta family, a crystal structure of the C1q domain of Cbln1, biophysical analysis of the molecular interactions within the Neurexin-Cbln1-GluD2 trans-synaptic complex, as well as evidence for the domain arrangement of the ecto-domain of the iGluR-Delta proteins. Together, these data enhance our knowledge of the molecular details of this macro-molecular complex and provide evidence to support models for the mechanisms of their involvement in synapse formation and function, thereby making a contribution to the vast and medically relevant field of molecular neurobiology.
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