Leung, Yeung Yeung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 134-154). / Abstracts in English and Chinese. / CONTENTS --- p.i / ACKNOWLEDGEMENTS --- p.iv / ABSTRACT --- p.v / 論文摘要 --- p.viii / LIST OF FIGURES --- p.x / LIST OF TABLES --- p.xiv / LIST OF ABBREVIATIONS --- p.xv / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 1.1 --- Brain iron function and diseases --- p.1 / Chapter 1.1.1 --- Function of iron in the brain --- p.1 / Chapter 1.1.2 --- Iron involved oxidative damage --- p.2 / Chapter 1.1.3 --- Role of iron in neurodegenerative diseases --- p.6 / Chapter 1.1.4 --- Role of iron in Alzheimer's disease --- p.7 / Chapter 1.1.5 --- Deleterious effects of iron in memory function --- p.9 / Chapter 1.2 --- Iron regulation in the brain --- p.10 / Chapter 1.2.1 --- Transport and storage of brain iron --- p.10 / Chapter 1.2.2 --- Iron homeostasis in the brain --- p.14 / Chapter 1.2.3 --- Transport of iron in axon and synapse --- p.17 / Chapter 1.3 --- The hippocampus --- p.19 / Chapter 1.3.1 --- Hippocampus and memory function --- p.19 / Chapter 1.3.2 --- Structure of the hippocampus --- p.20 / Chapter 1.3.3 --- Cell composition in the hippocampus --- p.26 / Chapter 1.3.4 --- Wiring in the hippocampus --- p.28 / Chapter 1.4 --- Synaptic plasticity and long term potentiation --- p.30 / Chapter 1.4.1 --- Basic theory of synaptic plasticity --- p.30 / Chapter 1.4.2 --- Types of synaptic plasticity --- p.30 / Chapter 1.4.3 --- The discovery of long term potentiation --- p.31 / Chapter 1.4.4 --- Long term potentiation --- p.32 / Chapter 1.4.5 --- Cellular mechanism of long term potentiation --- p.33 / Chapter 1.4.6 --- Role of reactive oxygen species in long term potentiation --- p.36 / Chapter 1.5 --- Aim of the study --- p.38 / Chapter 2. --- MATERIALS AND METHODS --- p.39 / Chapter 2.1 --- Rat model of iron overload --- p.39 / Chapter 2.2 --- Multi-electrode field potential measurement --- p.40 / Chapter 2.2.1 --- Acute preparation of hippocampal slices --- p.40 / Chapter 2.2.2 --- Multi-electrode array recording system --- p.41 / Chapter 2.2.3 --- Recording of field excitatory postsynaptic potentials --- p.42 / Chapter 2.2.4 --- Induction of LTP --- p.47 / Chapter 2.2.5 --- Recording of paired-pulse ratio --- p.48 / Chapter 2.3 --- Whole cell patch-clamp recordings --- p.50 / Chapter 2.4 --- Biochemical assays --- p.57 / Chapter 2.4.1 --- Preparation of brain homogenate --- p.57 / Chapter 2.4.2 --- Total iron measurement --- p.57 / Chapter 2.4.3 --- Protein carbonyl measurement --- p.58 / Chapter 2.4.4 --- Determination of reactive oxygen species --- p.60 / Chapter 2.5 --- Drugs and data analysis --- p.61 / Chapter 3. --- RESULTS --- p.62 / Chapter 3.1 --- The acute effects of extracellular iron on synaptic transmission and long-term synaptic plasticity in the hippocampus in vitro --- p.63 / Chapter 3.1.1 --- Effects of ferric ion on basal synaptic transmission --- p.63 / Chapter 3.1.1.1 --- Effect of FAC on basal fEPSPs --- p.63 / Chapter 3.1.1.2 --- Comparison with the effect of AC on basal fEPSPs --- p.69 / Chapter 3.1.2 --- Effects of ferric ion on long-term synaptic plasticity --- p.72 / Chapter 3.1.2.1 --- Effect of acute FAC treatment on LTP --- p.72 / Chapter 3.1.2.2 --- Comparison with the effect of AC on LTP --- p.75 / Chapter 3.1.3 --- Effects of ferric chloride --- p.78 / Chapter 3.1.4 --- Effects of ascorbic acid on the action of FAC --- p.81 / Chapter 3.2 --- "The acute, in vitro effect of extracellular iron on the membrane properties and excitability of hippocampal CA1 neurons" --- p.86 / Chapter 3.2.1 --- Membrane input resistance --- p.86 / Chapter 3.2.2 --- Voltage-Current relationship --- p.88 / Chapter 3.2.3 --- Membrane excitability --- p.90 / Chapter 3.2.3.1 --- Threshold current --- p.90 / Chapter 3.2.3.2 --- Action potential firing frequency --- p.92 / Chapter 3.2.4 --- Action potential characteristics --- p.95 / Chapter 3.2.4.1 --- "Action potential amplitude, area and width" --- p.95 / Chapter 3.2.4.2 --- Rise and decay kinetics of action potential --- p.98 / Chapter 3.3 --- The chronic effects of iron-loading in the brain on hippocampal long-term synaptic plasticity --- p.100 / Chapter 3.3.1 --- Validation of the iron-overload model --- p.100 / Chapter 3.3.1.1 --- Short-term (1 week) treatment --- p.100 / Chapter 3.3.1.2 --- Long-term (4 weeks) treatment --- p.103 / Chapter 3.3.2 --- Effects of chornic iron-overloading on LTP --- p.105 / Chapter 3.3.2.1 --- Short term iron treatment --- p.105 / Chapter 3.3.2.2 --- Long term iron treatment --- p.108 / Chapter 3.3.3 --- Oxidative stress measurement --- p.111 / Chapter 3.3.3.1 --- Protein oxidation --- p.111 / Chapter 3.3.3.2 --- Reactive oxidative species level --- p.116 / Chapter 4. --- DISCUSSION --- p.120 / Chapter 4.1 --- "Acute, in vitro effects" --- p.121 / Chapter 4.2 --- "Chronic, in vivo effects" --- p.125 / Chapter 5. --- REFERENCES --- p.134

Au niveau de la synapse, la liaison des neurotransmetteurs aux récepteurs membranaires induit l’ouverture de canaux ioniques. Le Récepteur de la Glycine (RGly) est un récepteur ionotrope impliqué dans des troubles neuronaux tels que l’addiction, la douleur chronique, ou l’hyperekplexie ; pour cette raison il est important de développer des nouveaux traitements ciblant ce récepteur. Nous avons utilisé des simulations de Dynamiques Moléculaire (DM) et d’électrophysiologie numérique afin d’évaluer la fonction des structures du RGly disponibles et montré qu’aucune d’entre elles ne satisfait les propriétés fonctionnelles de l’état ouvert. Grâce aux simulations de DM, nous avons caractérisé une nouvelle conformation du RGly, qui est compatible avec cet état. Nous avons souligné le rôle majeur des portails latéraux pour la perméation des ions. Nous avons proposé un protocole, nommé pharmacologie dépendante de l’état, pour identifier des molécules modulatrices de protéines allostériques. / Signals within neurons are mostly transmitted through chemical synapses. Signal transduction arises from the binding of neurotransmitters to membrane receptors in order to open ion channels. The Glycine Receptor (GlyR) is an ionotropic receptor which is involved in several neurological disorders such as addiction, chronic pain, or hyperekplexia. Because of its implication in human diseases, it is interesting to design novel drugs targeting this receptor. We used Molecular Dynamics (MD) simulations and computational electrophysiology to probe the function of available GlyR structures. We showed that none of the experimental structures display the physiological behavior of the conductive state. Using MD simulations, we captured a novel conformation of the GlyR compatible with a conductive state and demonstrated the importance of lateral portals for ionic permeation. Lastly, we proposed an original protocol, named state-based pharmacology, to discover modulators of allosteric proteins.

Synaptic transmission alters the strength of the postsynaptic potential, through a process called short-term synaptic plasticity (STP). In this study, endplate potentials (EPPs) from the frog neuromuscular junction were used to resolve and quantify the presynaptic components involved in enhancement and depression of transmitter release during repetitive stimulation under normal quantal release conditions (2 mM Ca2+, 1mM Mg2+). During trains of stimulation given between 10 - 200 Hz, the amplitude of the EPPs first increased then decreased; a maximum increase of 77% was produced after 2-4 stimuli. EPP amplitudes began to increase at ~ 20 Hz, were maximal at ~ 55 Hz, and thereafter, decreased as the rate of stimulation increased. The integrated total release after 25 stimuli was little changed across frequencies between 10 - 100 Hz. EPPs ran down in two phases: a fast phase, attributed to the depletion of a readily releasable pool (RRP) of synaptic vesicles, followed by a slow phase, attributed to the depletion of vesicles from a depot pool (DP). Depletion of the readily releasable pool of synaptic vesicles (RRP) was determined by quantifying release under the fast and slow time rundowns and subtracting the number of vesicles associated with mobilization to the RRP from the total number of vesicles released during stimulation trains of 50 impulses. Impulses were delivered at 12 different rates ranging from 50 to 200 /s. Estimates of the number of vesicles released from the RRP increased with frequency of stimulation until maximal depletion levels of 5500 - 6000 vesicles were reached at stimulation rates between 90-130/s, assuming a control quantal content of 200 vesicles released per impulse. Depletion was less at lower frequencies when the number of stimuli delivered was identical. When the RRP maximally depleted, release was inversely related to stimulation rate, as would be expected if mobilization from the depot pool was the sole determinate of release during the slow phase. An equation constructed from four known components of enhancement and two components of depression - the depletion of vesicles from a readily releasable pool (RRP) and from the depot pool (DP) that refills the RRP, was used to fit and then simulate EPPs obtained during trains using different patterns of stimulation and varying amounts of extracellular Ca2+; the decay time constant parameters of enhancement, numerically derived from the observed data, were fixed at tau ~ 46, 220, 1600, and 20000 ms. The number of components of enhancement necessary to approximate the data decreased, from four in low (0.14 - 0.2mM) extracellular Ca2+, to one (tau ~ 46 ms) in 2.0 mM extracellular Ca2+, but four components of enhancement were necessary to fit the data when the amplitude of the EPP was not depressed below the control amplitude. This model was able to predict within ~ 3 % EPP amplitudes over a 10-fold range of frequency and Ca2+ concentration.

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Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 200-233). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;