1,721,001 research outputs found
Focusing light in biological tissue through a multimode optical fiber: refractive index matching
Full text linkControlling light propagation through a step-index multimode optical fiber (MMF) has several important applications, including biological imaging. However, little consideration has been given to the coupling of fiber and tissue optics. In this Letter, we characterized the effects of tissue-induced light distortions, in particular those arising from a mismatch in the refractive index of the pre-imaging calibration and biological media. By performing the calibration in a medium matching the refractive index of the brain, optimal focusing ability was achieved, as well as a gain in focus uniformity within the field-of-view. These changes in illumination resulted in a 30% improvement in spatial resolution and intensity in fluorescence images of beads and live brain tissue. Beyond refractive index matching, our results demonstrate that sample-induced aberrations can severely deteriorate images from MMF-based systems
Investigations on synaptic plasticity: new tools and perspectives
Full text linkFor decades, neuroscientists have sought to understand the neural basis of learning and memory. As yet no unified model has emerged, although it is widely agreed that synaptic plasticity plays a pivotal role. In consequence, a great deal of research has focused upon the characterisation of synaptic plasticity both in vitro and in vivo. While the advances have been impressive, substantial gaps remain that may be slowing the identification of the precise relationship between synaptic plasticity and learning and memory: (1) there is a paucity of research examining presynaptic plasticity mechanisms and presynaptic receptor functions; (2) synaptic dynamics are studied in behaving animals without consideration of the impact of endogenous factors, even though it looks to be clear that hormone signalling impacts upon synaptic processes: (3) An accurate assessment of synapse biology with respect to cognitive function can only arise from studies in living animals, an ambition that will require the development of new imaging tools. In this thesis I begin to address each of these points. First, using a combination of optical and electrophysiological tools I provide a functional framework for presynaptic NMDA receptor signalling that has been previously lacking. I show that the specific subunit expression at presynaptic terminals is important as it regulates Ca2+ dynamics. Second, I demonstrate that oestrogen, a neuromodulator, lowers the threshold of plasticity in several brain regions, emphasising how important it is to consider hormone status when studying synaptic dynamics. Finally, I implement several advances to a multimode fibre based minimally invasive system for high-resolution brain imaging, including the first examples of two-photon MMF imaging of biological tissue and successive imaging cycles through a multimode fibre
Mechanisms of long-term presynaptic plasticity at Schaffer-collateral synapses
Full text linkSynaptic plasticity is thought to be integral to learning and memory. The two most common forms of plasticity are long-term potentiation (LTP) and long-term depression (LTD), both of which can be supported either by presynaptic changes in transmitter release probability (Pr), or by postsynaptic changes in AMPA receptor number. It is generally thought that the induction of LTP and LTD at Schaffer-collateral synapses in the hippocampus depends on the activation of NMDA receptors (GluN). Recent studies, however, have demonstrated that both increases and decreases in Pr can be induced under blockade of postsynaptic GluN receptors, suggesting that the activation of postsynaptic GluN receptors by glutamate is only a strict requirement for postsynaptic plasticity. In this thesis, I therefore re-examined the role of glutamate in presynaptic plasticity. I used single synapse imaging along with electrophysiological and pharmacological techniques to independently manipulate and monitor the levels of glutamatergic signalling during synaptic activity. I discovered that glutamate is inhibitory and unnecessary for the induction of LTP at the presynaptic locus. My findings support a novel model of presynaptic plasticity in which the net activity-dependent changes in Pr at an active presynaptic terminal is jointly determined by two opposing processes that can be simultaneously active: 1) postsynaptic depolarization, which, via the activation of L-type voltage-gated Ca2+ channels, increases Pr by driving the synthesis and release of nitric oxide from neuronal dendrites and 2) glutamate release, which through the activation of presynaptic GluN receptors, decreases Pr. Computationally, this model suggests that plasticity functions to reduce prediction-errors that arise during synaptic activity, and, thereby offers a biologically plausible mechanism by which neuronal networks may optimize learning at the level of single synapses.</p
Burnout in medicine: A novel approach exploring the impact of uncertainty and the use of biomarkers as a measurement tool
Full text linkBurnout constitutes a significant challenge for healthcare organisations not only in respect to individual wellbeing but also in terms of the catastrophic downstream implications to patient safety, patient satisfaction, and quality of care. This DPhil, divided into three parts, aims to explore burnout in healthcare professionals, an accelerating phenomenon that is hotly discussed but minimally understood, with a focus on the impact uncertainty has and an exploration into the novel use of neurohormones as potential biomarkers of wellbeing.
Part I: Exploration of concepts: burnout and uncertainty.
Chapter 1 presents a selective overview of the broad context of burnout in healthcare, reporting existing literature on the impact of burnout—to physicians, to patients, and to healthcare organisations—and considering the challenges that pertain to burnout research, particularly relating to challenges in measurement. Chapter 2 explores the presence of uncertainty in the healthcare environment looking at what impacts an individual’s tolerance of uncertainty and how the reaction affects provider- and patient-centred outcomes. Chapters 3 and 4 present two observational studies exploring drivers of satisfaction at work for faculty in an academic medical centre, with an analysis of the interplay between burnout and uncertainty in the clinical environment. Chapter 5 presents a study that looks at how language used in clinical hand-over affects sense of uncertainty in the receiving clinician, demonstrating how language variation can influence emotional perception of uncertainty.
Part II: Exploration of biology: exploration into use of biomarkers to measure burnout.
Chapters 6 and 7 explore the novel use of cortisol and oxytocin levels as potential biomarkers of stress and burnout in clinical faculty at a large teaching hospital.
Part III: Exploration of interventions to reduce burnout and strategies to embrace uncertainty.
Chapter 8 presents an interprofessional intervention study looking to reduce burnout through self-facilitated groups meeting monthly for three months. This study re-affirms the importance of the findings presented in this thesis and points to the need for more interventions aimed at enhancing trainee and faculty wellbeing. Chapter 9 synthesises current literature on tolerance of uncertainty alongside findings of the thesis, self-experience, and experience of colleagues and students in a narrative review to identify strategies to help clinicians thrive in the face of clinical uncertainty.
Finally, Chapter 10 presents an overview of the key findings from each study, their methodological strengths and limitations, directions for future research, and implications for clinical training, the measurement of wellbeing initiatives, and patient care
Surface-modified microparticles of iron oxide: biodistribution, systemic inflammatory modulation and MRI applications in the early stages of CNS inflammation
Full text linkAn investigation of presynaptic plasticity mechanisms
Full text linkThe regulation of synaptic strength is thought to underlie the complex emergent dynamics of neural networks. The strength of a synapse is determined by its pre- and postsynaptic properties, both of which are under tight regulatory control orchestrated by ongoing neuronal activity. We now have a good understanding of the plasticity rules underlying the regulation of postsynaptic strength. The same set of rules has been imposed onto the regulation of presynaptic strength. However, the operation of pre- and postsynaptic terminals is fundamentally different, both on a mechanistic and functional level. I have therefore systematically investigated the mechanisms underlying presynaptic plasticity at the Schaffer collateral-CA1 synapse under varying conditions of synaptic activity. I have examined three general modes of regulation: (1) synaptic changes dependent on the concerted activity of pre- and postsynaptic neurone (homosynaptic plasticity); (2) changes dependent only on presynaptic activity; (3) changes dependent on postsynaptic activity alone (heterosynaptic plasticity). Using a combination of electrophysiological and optical techniques, I have monitored and manipulated the strengths of single synapses both pre- and postsynaptically, which allowed me to impose certain activity patterns and investigate resulting synaptic modifications. I found that heterosynaptic plasticity along local segments of dendrites is expressed at both synaptic loci and depends on the spatial arrangement of synapses. Pre- and postsynaptic strength changes were weakly correlated and pharmacologically dissociable. Next, I found that glutamate release suppresses both short- and long-term presynaptic function, which required the activation of presynaptic NMDA receptors. Lastly, I found that presynaptic long-term potentiation (LTP) is spike timing-dependent but does not rely on coincidence detection via postsynaptic NMDA receptors. My findings suggest that the presynaptic terminal is functionally distinct, which is reflected in parallel regulatory pathways. I suggest the synapse to be viewed as a two- compartment model, consisting of a presynaptic non-linear transformation followed by postsynaptic linear weighting
Consequences of presynaptic dysfunction in Alzheimer’s Disease models
Full text linkSoluble oligomeric assemblies of amyloid β (Aβo) drive much of Alzheimer’s disease (AD) pathology. Among these, changes in synaptic plasticity including attenuated long-term potentiation (LTP), and facilitated long-term depression (LTD), accompanied by postsynaptic weakening, are believed to be significant contributors to disease progression. Much work has been directed at characterising the postsynaptic effects of oligomers, with little or conflicting evidence of their impact at the presynaptic bouton. Some studies investigating presynaptic boutons directly, have suggested that Aβo elevate presynaptic transmitter release. Given that transmitter release precedes postsynaptic processes, an alteration in presynaptic biology could drive postsynaptic pathology. One important downstream process of AD pathology is tau hyperphosphorylation. The relationship between Aβo and tau phosphorylation is unknown. Recent evidence that tau plays a physiological role in AMPAR endocytosis as a result of LTD induction might provide a link between tau phosphorylation and Aβo-induced synaptic changes. Here I probe the presynaptic effects of Aβo and how they impact AD pathogenesis. I confirm that Aβo can enhance both evoked, and non-evoked spontaneous miniature presynaptic release using direct, optical techniques in rat hippocampal cultures. Furthermore, I show that partial reduction in presynaptic function can restore Aβo-induced plasticity deficits in adult mouse acute hippocampal slices. I also demonstrate that these plasticity deficits might underly tau pathology; In rat organotypic slices I show that chronic NMDAR-dependent LTD can cause pathological tau phosphorylation, mimicking the effects of chronic Aβo incubation, which also requires NMDARs. In addition, I show that reduction in presynaptic function prevents Aβo-induced pathological tau phosphorylation, suggesting that the pathological tau phosphorylation that occurs in AD could be a result of repeated LTD-inducing conditions driven by an enhancement of presynaptic release probability. Previously, little work has been done to establish a role for presynaptic changes in AD. I have developed this area within the thesis and have established a link between aberrant Aβo aggregation and pathological tau phosphorylation that could have an important impact on AD therapy
Structural plasticity and network dynamics in the dorsal hippocampus - accumbal circuitry
Full text linkThe dorsal hippocampus has been implicated in the encoding of contextual information with particular involvement in spatial navigation. The nucleus accumbens is thought to promote the translation of higher order cognitive modalities into motor function directives. The dorsal hippocampus-accumbal circuitry has been postulated to promote the acquisition, consolidation and reinstatement of context specific reward seeking behaviour. However, the corresponding anatomical connectivity and physiological underpinnings of the translation of spatial representations into behavioural directives have not yet been established. This thesis entails the first study of the hippocampal dCA1-accumbal pathway whereby two complementary approaches were employed. Synaptic plasticity mechanisms, believed to underly experience dependent functional adaptations in the hippocampus and nucleus accumbens, have been shown to correlate with structural changes in dendritic spine morphology. For the sake of their study, invasive techniques have been established, however have been shown to incur significant damage to associated circuitries, rendering insights into structural plasticity in deep brain regions challenging. This thesis entails the first full in vitro and in vivo proof of principle of a new approach toward minimally invasive high resolution deep brain imaging of dendritic spines utilising adaptive optics and holographic light propagation through a single optical fibre, thinner than a human hair. This technology reduces the footprint and thus resulting tissue damage by two orders of magnitude while maintaining diffraction limited resolution. This work might facilitate future investigations of structural synaptic plasticity in deep brain circuitries such as the hippocampal dCA1-accumbal pathway (supervisor: Prof. Dr. Nigel Emptage). There has been indication that spatial information guided behaviour might be dependent on a functionally intact integrated dorsal hippocampus - accumbal circuitry. In order to study the downstream effect of dorsal hippocampal spatial information processing on the network dynamics of the accumbal circuitry, large scale multichannel electrophysiological recordings combined with optogenetic stimulation were employed simultaneously in both structures. dCA1 pyramidal cell long range projections were shown to mono-synaptically drive the activity of parvalbumin expressing interneurones in the NAc which in turn orchestrate the activity of medium spiny neurons through feed forward inhibition in a context dependent manner. This facilitates the selective activity of discrete medium spiny neuron cohorts and supports the translation of neural representations of space into spatial appetitive behaviour. Furthermore, this work might help to conceptualise how the nucleus accumbens can act as a gateway for converging and potentially competing input from different corticolimbic systems (supervisor: Ass. Prof. Dr. David Dupret).</p
An exploration of the impact of amyloid-β on intracellular Ca2+ signalling and metabolic pathways in models of Alzheimer’s Disease
Full text linkIn the field of Alzheimer’s Disease (AD) research, there’s a growing consensus that therapeutic intervention to significantly modify disease progression must begin at the preclinical stages of AD. By investigating early disease mechanisms, researchers can develop targeted therapies aimed at preventing the onset of cognitive symptoms. The amyloid hypothesis of AD suggests that the accumulation of Aβ peptides, particularly Aβ oligomers, is a central and early event in AD pathogenesis. Aβ oligomers are neurotoxic and cause synaptic dysfunction, eventually resulting in synapse loss and neuronal death, which contributes to the cognitive decline observed in AD patients. The mixed results observed in clinical trials of antibodies targeting Aβ highlight the necessity of identifying alternative molecular targets for AD therapeutics. Furthermore, the exact role of Aβ in AD pathophysiology is still under constant investigation. Extensive research efforts have focused on characterising the effects of Aβ oligomers at the pre- and postsynaptic compartment, intracellular Ca2+ signalling, and structural plasticity of dendritic spines. Although, there are still significant gaps in our understanding, which may delay the comprehension of the relationship between early disease mechanisms and late-stage cognitive decline in AD. In this thesis, I aim to advance the understanding of the role that Aβ oligomers play in synaptic function and Ca2+ signalling mechanisms in CA1 hippocampal neurons by using synthetic Aβ oligomers applied to hippocampal neurons and slice cultures, as well as in acute hippocampal slices of J20 mice, an AD model. Using a combination of electrophysiology and imaging techniques, I firstly investigated the effects of a presynaptic Ca2+ channel, Cav2.1, in mediating Aβ oligomer toxicity. A heterozygous knockout of Cav2.1 normalised presynaptic function and rescued Aβ-induced LTP impairment, while preserving basal neurotransmission. Secondly, I assessed lysosomal and endoplasmic reticulum (ER) activity-dependent dynamics and Ca2+ release, and the effects on structural plasticity in response to Aβ oligomer treatment. Aβ oligomers were able to disrupt specific features of lysosome dynamics and recruit Ca2+-induced Ca2+ release (CICR) from the ER in response to back-propagating action potentials in neuronal dendrites. Finally, 1H Nuclear Magnetic Resonance (NMR) metabolomics was used to identify metabolite changes throughout regular aging and AD pathogenesis in tissues from wild type and J20 mice. Both regular aging and early/late stage AD induced specific metabolic signatures, which may contribute to the discovery of novel biomarkers associated with metabolites altered in AD
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