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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 effects of cannabinoid agonists on spontaneous neuronal network activity were characterized in murine spinal cord and auditory cortical cultures with multichannel extracellular recording using photoetched electrode arrays. Different cultures responded reproducibly with global decreases of spiking and bursting to anandamide and methanandamide, but each agonist showed unique minor effects on network activity. The two tissues responded in a tissue-specific manner. Spontaneous activity in spinal tissue was terminated by 1 μM anandamide and 6.1 μM methanandamide. Cortical activity ceased at 3.5 μM and 2.8 μM respectively. Irreversible cessation of activity was observed beyond 8 μM for both tissues and test substances. Palmitoylethanolamide, demonstrated that CB2 receptors were not present or not responsive. However, the data strongly suggested the presence of CB1 receptors.

Cell-cell signalling molecules play important roles in neural development. In response to extracellular signals, neuronal progenitor cells proliferate, differentiate, and form a neuronal network. In the vertebrate retina, retinal ganglion cells (RGCs) are the first neurons produced during development and are the only neurons that send projections to the brain. However, the molecular mechanisms for RGC development have not been fully understood. In this study, I have investigated the expression and functions of Nel (Neural Epidermal Growth Factor Like), an extracellular glycoprotein that contains chordin-like domains and epidermal growth factor-like domains, in the development of the chick RGCs and retinotectal projection. I found that on embryonic days (E) 2-3.5, Nel was expressed in the presumptive retinal pigment epithelium of the developing eye. Correspondingly, Nel-binding activity (Nel receptor activity) was detected in the retinal pigment epithelium and also the progenitor layer of the neural retina. At the early stages during RGC formation, Nel overexpression increased the total number of RGCs and accelerated the progression of RGC differentiation wave. Conversely, Nel expression knockdown decreased the total number of RGCs and slowed down the progression of RGC differentiation wave. At later stages (E3-E18), expression of Nel in the retina was in the retinal pigment epithelium and the RGC layer, whereas receptor activity for Nel was localized in the retinal pigment epithelium and the RGC axons. In vivo, Nel overexpression in the developing retina induced the inhibition of RGC axons and thus disrupting the intraretinal RGC axon projection. These results suggest that Nel can positively regulate the production of RGCs at the early stages during retinal development, and at the later stages, Nel can function as an inhibitory guidance cue in vivo for RGC axons.

The present work investigates the applicability of Genetic Algorithms (GA) to the problem of signal propagation in Native Neuronal Networks (NNNs). These networks are comprised of neurons, some of which receive input signals. The signals propagate though the network by transmission between neurons. The research focuses on the regeneration of the output signal of the network without knowing the original input signal. The computational complexity of the problem is prohibitive for the exact computation. We propose to use a heuristic approach called Genetic Algorithm. Three algorithms are developed, based on the GA technique. The developed algorithms are tested on two different networks with varying input signals. The results obtained from the testing indicate significantly better performance of the developed algorithms compared to the Uniform Random Search (URS) technique, which is used as a control group. The importance of the research is in the demonstration of the ability of GA-based algorithms to successfully solve the problem at hand.

The present work investigates the applicability of Genetic Algorithms (GA) to the problem of signal propagation in Native Neuronal Networks (NNNs). These networks are comprised of neurons, some of which receive input signals. The signals propagate though the network by transmission between neurons. The research focuses on the regeneration of the output signal of the network without knowing the original input signal. The computational complexity of the problem is prohibitive for the exact computation. We propose to use a heuristic approach called Genetic Algorithm. Three algorithms are developed, based on the GA technique. The developed algorithms are tested on two different networks with varying input signals. The results obtained from the testing indicate significantly better performance of the developed algorithms compared to the Uniform Random Search (URS) technique, which is used as a control group. The importance of the research is in the demonstration of the ability of GA-based algorithms to successfully solve the problem at hand.

Spontaneously active frontal cortex and spinal cord networks grown on microelectrode arrays were used to study effects of D-methylphenidate. These central nervous system tissues have relatively low concentrations of dopaminergic and noradrenergic neurons compared to the richly populated loci, yet exhibit similar neurophysiological responses to methylphenidate. The spontaneous spike activity of both tissues was inhibited in a concentration-dependent manner by serial additions of 1-500 µM methylphenidate. Methylphenidate is non-toxic as spike inhibition was recovered following washes. The average concentrations for 50% spike rate inhibition (IC50 ± SD) were 118 ± 52 (n= 6) and 57 ± 43 (n = 11) for frontal cortex and spinal cord networks, respectively. A 3 hour exposure of a network to 1 mM methylphenidate was nontoxic. The effective concentrations described in this study are within the therapeutic dosage range. Therefore, the platform may be used for further investigations of drug mechanisms.

An Active Microscaffold System with Fluid Delivery and Stimulation/Recording Functionalities for Culturing 3-D Neuronal Networks Laura Elizabeth Rowe 215 Pages Directed by Dr. A. Bruno Frazier An active microscaffold system with fluid delivery and electrical stimulation/recording functionalities for 3-D neuronal culture studies is presented. The microscaffolds presented in this dissertation consist of an array of microfabricated towers with integrated microfluidic channels, fluid ports, and electrodes. The microfluidic channels and ports allow for perfusion of nutrients, gas exchange, and biochemical control of the extracellular environment throughout the 3-D culture, while the electrodes allow for active stimulation/recording of the 3-D neuronal network. In essence, the microscaffold serves as an artificial circulatory system to enable 3-D in vitro growth and proliferation of re-aggregate neuronal cell cultures. Increased cell survival on microscaffolds with nutrient perfusion at 14 and 21 days in vitro (DIV) is presented. Additionally, the microtower scaffold is built upon a substrate that is compatible with the Multi Channel Systems preamplifier setup to enable electrical stimulation/recording of the cultured network in a 3-D mutilelectrode array (MEA) environment. Impedance measurements on the functioning microtower electrodes were obtained. The overall goal of this research was to develop a BioMEMS technology to provide neuroscientists with a better investigative tool for studying 3-D in vitro neuronal networks than is currently available.

Spontaneously active monolayer neuronal networks cultured on photoetched multimicroelectrode plates (MMEPs) offer great potential for use in studying neuronal networks. However, there are many problems associated with frequent, long-term use of MMEPs. The major problems include (1) polysiloxane insulation deterioration and breakdown, (2) and loss of gold at the gold electroplated indium-tin oxide (ITO) electrodes. The objective of this investigation was to correct these major problems. Quality control measures were employed to monitor MMEP fabrication variables. The phenotypes of polysiloxane degradation were identified and classified. Factors that were found to contribute most to insulation deterioration were (1) moisture contamination during MMEP insulation, (2) loss of the quartz barrier layer from excessive exposure to basic solutions, and (3) repetitive use in culture. As a result, the insulation equipment and methods were modified to control moisture-dependent insulation deterioration, and the KOH reprocessing solution was replaced with tetramethylguanidine to prevent damage to the quartz. The problems associated with gold electroplating were solved via the addition of a pulsed-DC application of gold in a new citrate buffered electroplating solution.