Neuronal death due to excitotoxicity and oxidative stress is a critical part of several major disease processes, including ischaemic brain damage and Alzheimer’s disease. This study used cultures of cerebellar granule neurones as a model for investigation of these processes, considering both their pharmacological and molecular aspects. Another important part of the study was the development and investigation of a mode of neuroprotection which was effective in protecting against these factors. Additionally, the optimal culturing conditions for neurone survival were determined and the efficacy of two cell viability assays established. Examination of excitotoxicity and oxidative stress considered the effects of glutamate, N-methyl-D-aspartate, 3-nitropropionic acid and oxygen-glucose deprivation. Additionally, extensive study was carried out of the actions of increased glucose concentration and a range of metabolites of the essential amino acid tryptophan. It was demonstrated that several tryptophan metabolites induced neurotoxic effects, including 5-hydroxyanthranilic acid, which caused neuronal death via oxidative damage mediated by generation of reactive oxygen species and prevented by catalase but not superoxide dismutase. 5-Hydroxyanthranilic acid treatment also led to activation of the p38 signalling pathway, although the cell death caused was independent of caspase-3 activation. Investigation of neuroprotection was concerned with establishing an effective method of protection, with a range of stimuli used to precondition neurones, such as N-methyl-D-aspartate, 3-nitropropionic acid, hydrogen peroxide, bicuculline and 4-aminopyridine. Preconditioning with 100µM N-methyl-D-aspartate at 8 DIV was effective in protecting against the neurotoxic effects of glutamate or 3-nitropropionic acid applied 24 hours after the commencement of preconditioning, but was not effective against oxygen-glucose deprivation of 4 hours duration. Preconditioning with 2.5mM 4-aminopyridine was effective in providing protection against a range of insults (glutamate, N-methyl-D-aspartate, 3-nitropropionic acid). This protection was independent of N-methyl-D-aspartate receptor activation, reduced by blockade of depolarisation and was effective in protecting against caspase-3-independent cell death.

Cannabinoids have well known immunomodulatory properties, through actions mainly on CB2 receptors, which are abundantly expressed in microglia, and or other yet as unidentified receptors. The primary objective of this study, therefore, was to investigate the possibility of modulating particular microglial functions (nitric oxide (NO) release, endocannabinoid hydrolysis and calcium mobilisation) with cannabinoids using an immortalized cell lines, specifically, mouse-derived BV -2 cells and rat-derived Highly Aggressive Proliferating Immortalized (HAPI) cells to overcome the challenges of having enough primary microglia for experimental use. NO release was stimulated with lipopolysaccharide (LPS) and or interferon y (IFN-y) and quantified using the Greiss reaction. While the selective CB2 agonist, JWH133, a number of endocannabinoids and the CBI and CB2 receptor antagonists, rimonabant and SR144528, all failed to significantly alter LPS-stimulated NO formation in RAPI or BV -2 cells, the non-selective CBI/CB2 agonist CP55940 (only at 10 ~M) significantly attenuated NO formation in both HAPI and BV -2 cells. Medium from ischaemic neuroblastoma cells, SHSY5Y cells, and the PPARy agonist, rosiglitazone, were also able to attenuate LPS-induced NO release via CB2 receptor-independent mechanisms. Although IFN-y enhanced LPS-induced NO release in BV-2 cells without affecting basal levels, WIN55-212-2 and JWH133 ( 1 uM) were unable to affect LPS/IFN-y induced NO release. HAPI and BV-2 cells appeared to express CB] and CB2 receptor protein using immunoblotting, but RT-PCR revealed a lack of mRNA expression of either receptor in both cell lines. Endocannabinoid, AEA and PEA, hydrolysis was assessed in BV-2 cells by monitoring liberation of [3H]-ethanolamine from labelled AEA or PEA. In intact BV -2 cells, marked differences in the time courses for PEA and AEA hydrolysis were observed, raising the possibility of a distinct microglial enzyme responsible for PEA hydrolysis. Expression of GPR55, in BV-2 cells was confirmed using qRT-PCR analysis. LPI, evoked calcium elevations in a concentration-dependent manner. Although cannabinoids were unable to produce a significant calcium response in BV-2 cells, CBD and i19-THC produced attenuations of LPI-induced calcium elevation while CP55940 and rimonabant, showed less significant reductions. In order to evaluate the role of GPR55, two batches of GPR55- HEK293 cells from different sources were employed; however neither appeared to express GPR55 mRNA. CBD and ∆9-THC were also able to attenuate calcium responses to the P2 receptor agonists, ATP and ADP, in BV- 2 cells indicating a non-selective antagonist activity, as well as the possibility of P2_ receptor expression in BV -2 cells. Although GPR55 agonists were unable to stimulate microglial migration in an in vitro assay, ATP (100 u M) significantly enhanced microglial migration which was inhibited by both CBD and ∆9 - THC (10uM). In conclusion, in the absence of conventional cannabinoid receptor expression in microglial cell lines, cannabinoids appear to affect microglia function through acting on the potential cannabinoid-like receptor, GPR55, and through P2 receptors, both of which may present new avenues in the treatment of neurodegenerative and neuroinflammatory illnesses.

Cultures of living cortical neurones, grown in vitro, provide a model of brain function, their longevity plus their openness to experimental manipulation and detailed observation, makes them ideally suited to investigations of micro-scale neural interactions. Functional connectivity between neural units can be defined according to the correlation of their activity, and the functional networks of mature cultured neurones have revealed a complex network organisation. Sharing many properties with other complex networks, such as the Internet, social networks and in vivo brain networks, cultures allow the investigation of how such a network emerges in a biological system. Moreover, they allow one to investigate the influence of experimental manipulation on shaping the network properties. However, very little work compares cultures' complex network properties from different experimental conditions and many questions remain unanswered. The aim of this thesis was to investigate how the functional connectivity of cultured neurones changes under different experimental conditions. A novel toolset and protocol was developed for analysing cultures' functional connectivity within a complex network framework. Spontaneously occurring and experimentally induced changes in connectivity were quantified by comparing topological, spatial and performance properties. Robust results followed from consideration of network topology, size and density influences on the complex network properties. Results confirmed that mature culture's networks are complex, and demonstrated that these emerge from an initially random network organisation to one that supports efficient network-wide flow of information. The influence of electrical stimulation on shaping the network properties is investigated with chronic stimulation during culture development. In conclusion, one can compare complex functional network properties from multiple experimental conditions by applying a reliable link definition, and accounting for differences in network size, density and topology. The present work reveals that the functional networks of cultured neurones spontaneously evolve to a topology optimised for information processing.

The δ-opioid receptor (DOR) shows potential as a therapeutic target for analgesia. DOR-targeting pharmaceutics may lead to fewer side effects than conventional opioid drugs such as morphine due to relatively low expression of DOR in the central nervous system (compared to the main target for morphine, the u-opioid receptor). The analgesic efficacy of DOR agonists increases following inflammation and receptor insertion has been suggested as a possible mechanism for this. Modulation of membrane expression of functional DOR receptors may be useful in the development of analgesic drugs. Currently, expression of functional DOR at the membrane of sensory neurons is controversial. Here, patch-clamp recordings and total internal reflection fluorescence (TIRF) microscopy have been used to study functional expression and trafficking of DOR in sensory neurons from rat trigeminal ganglia (TG). In addition, the role of inflammatory mediator bradykinin (BK) in DOR membrane expression has been investigated. To determine whether neurons express functional DCR, inhibition of voltage-gated Ca2+ channels (VGCC) by DCR agonist [D-Ala2, D- Leu5]-Enkephalin (DADLE) was determined. DADLE inhibited VGCC in 23% of TG neurons by 25.3 ± 5%. Pre-treatment with BK increased the population of DCR-positive neurons to 54%, but did not significantly affect the degree of VGCC inhibition by DADLE. Real-time TIRF microscopy revealed that BK treatment caused robust trafficking of DCR to the plasma membrane in neurons transfected with GFP-tagged DOR. In contrast, DADLE and TRPV1 agonist capsaicin caused a decrease in membrane abundance of DOR, suggesting internalisation of the receptor. In Ca2+ imaging experiments 80% of cultured TG neurons responded to BK, thus, these data suggest that a majority of BK-responsive TG neurons have the potential to become DOR-positive upon inflammation, re-establishing the therapeutic potential of peripheral DOR. In a separate line of investigation, enhancement of T-type VGCC in nociceptive neurons by BK has been discovered.

Dopaminergic (DA) neurons exhibit a short-latency, phasic response to unexpected biologically salient stimuli, including rewards. Despite extensive research on this DA signal, very little is known about the sources of sensory information reaching DA neurons. Previous research has identified the superior colliculus (SC) as the primary, if not exclusive route of short latency visual input to DA neurons. However, more recent research has suggested that the phasic DA response comprises two components; a short latency (50-110 ms), stimulus insensitive component, and a longer latency component (110-260 ms) that can reflect complex stimulus characteristics including reward value – more complex than might arise from intrinsic collicular processing. A solution to this apparent paradox may be suggested by recent studies that have demonstrated longer latency colour related responses in SC neurons. As the SC does not receive direct retinal input from colour sensitive cells, but it does receive input from a wide range of cortical structures, it is possible that cortical activation might underlie longer latency responses in the SC, which may in turn underlie longer latency responses in DA neurons. The aim of the research presented in this thesis was to investigate whether the cortex was capable of modulating the activity of DA neurons, and whether the SC was the relay for this cortical influence. In the anaesthetised rat, single pulse electrical stimulation of the barrel field of the primary somatosensory cortex (S1Bf) produced a short latency, short duration response in the SC, but DA neurons were largely insensitive to the stimulus. After disinhibition of the SC with the GABAA antagonist bicuculline, responses in the SC to S1Bf stimulation were enhanced, and DA neurons became responsive to S1Bf stimulation, suggesting that the SC is the route of cortical input to DA neurons. This was confirmed in the subsequent experiment. Responses were produced in DA neurons without the need for SC disinhibition by stimulating S1Bf with a high frequency train of pulses. This response in DA neurons was suppressed or eliminated by suppressing SC activity. Finally, the contribution of cortical and subcortical input to DA neuron responses was examined by stimulating the trigeminal nucleus. Trigeminal stimulation produced responses in the SC comparable to multiwhisker deflection, and produced responses in almost all DA neurons. Disinhibition of the SC differentially modulated phases of the SC response previously demonstrated to be produced by trigeminal and cortical input, and differential changes were seen in initial and later components of DA neuron responses, which were often associated with changes in the SC response. The results of these studies suggest that cortical inputs to the SC could provide a mechanism through which responses are produced in DA neurons that can reflect complex stimulus attributes. However, research in this thesis and elsewhere suggests that the activity of DA neurons is insufficiently discriminatory to reflect the full range of potentially rewarding stimuli, and hence it is suggested that DA neurons provide a salience signal, which can be biased by a pre-saccadic estimate of previously established reward value, but which does not communicate reward value per se.

In the last few decades, evidence has been accumulating for a role for xanthine oxidoreductase (XOR)-generated toxic reactive oxygen species (ROS) in a variety of pathological conditions that affect different organ systems. This enzyme in mammals exists in two inter-convertible forms: xanthine dehydrogenase (XDH) (the predominant intracellular form under physiological conditions) and xanthine oxidase (XO). A combination of XO and its oxidizable substrate xanthine (X) (or hypoxanthine (HX)) is widely used as a model to produce ROS and to study their effects in a variety of cell culture studies. However, the effect of the combination of XOR and the reduced nicotinamide adenine dinucleotide (NADH) in cell cultures is much less studied. NADH is another oxidizable substrate for XOR that binds to a different site on the enzyme from that of X binding. The aim of this project was to investigate some aspects of the in vitro toxicity of XOR, which might provide more insights into its in vivo toxicity. The main investigation was a comparison between the well studied X / XO and the much less studied NADH / XO toxicity models. Also, secondary studies were undertaken to investigate those aspects of X / XO toxicity where there are uncertainties about them. These studies were performed using primary cell cultures. Cell cultures are now widely used to study different diseases, and although they have their drawbacks, they have their advantages over the in vivo studies. For this project, primary cultures of cerebellar granule neurons (CGNs) were used. In the beginning, some problems were encountered with CGNs. The main problem was the immediate damage induced to the neurons (including those in the control groups) at the intervention/experiments day (i.e. day 8 or 9 after plating) by manipulating the cultures (i.e. aspirating the culture medium, adding treatment and control vehicles, and adding the restoration medium). After several months of investigation, it was serendipitously discovered that the immediate damage seen in the neurons (including those in the control groups) when they are manipulated at the experiments/intervention day was due to glutamate excitotoxicity (through activating its N-methyl-D-aspartate (NMDA) receptors). The source of glutamate was the fresh serum which is present at 10% V/V in the fresh culture medium that is added to the cultures at that day. After solving this problem, it was possible to conduct reliable experiments to investigate XO toxicity models. Regarding investigating XO toxicity, it was found that both of the X / XO and NADH / XO combinations were toxic to cultures of CGNs. However, the concentration of NADH needed to cause the toxicity was much higher than that of the other substrate, X, which is in agreement with previous cell-free experiments that showed that NADH is a much weaker substrate than X for the bovine milk XO used here. Blocking the site of X binding on XO prevented X / XO toxicity, but did not prevent NADH / XO toxicity. On the other hand, blocking the site of NADH binding prevented both X / XO and NADH /XO toxicities. Another difference between the two systems was that deactivating either superoxide or hydrogen peroxide (both are ROS) generated by XO prevented NADH / XO toxicity, whereas although deactivating hydrogen peroxide prevented X / XO toxicity, deactivating superoxide generated from this combination did not. In the NADH / XO system, an extracellular metal contaminant (likely contaminating XO powder/preparation) seemed to be involved in the toxicity. The two toxicity models were similar in the mediation of toxicity by intracellular iron ion. In X / XO toxicity, although superoxide generated extracellularly from the combination has no role in the toxicity, intracellularly produced superoxide seemed to play a role. Conclusions: 1. Culturing/experimental conditions have been optimised for viability studies in CGNs cultures. 2. The combination of NADH and XO induces damage to CGNs, where although blocking the NADH binding site prevents this damage, blocking the X binding site does not. It is feasible that the oxidation of NADH by some forms of XOR (other than the one used here) that are known to be very efficient in oxidizing NADH might produce in vivo toxicity. 3. A possibility raised by this study is that a metal (like the metal contaminant proposed to play a role in NADH / XO toxicity in this study) might contribute to XOR toxicity in vivo. 4. Intracellular superoxide often mediates XOR toxicity. 5. The results add support to many previous studies which suggested that intracellular hydroxyl radical (or a similar species) is involved in XOR toxicity.

Axons are directed along stereotypic pathways to their targets by cues arrayed in the extracellular environment. Identifying the cellular and molecular nature of these signals is of high interest and the developing optic pathway is a useful model system for achieving this. Although previous studies have identified several molecules essential for optic pathway formation, in vivo only subsets of retinal axons rely on them. I focused on the Dscam (Down’s syndrome cell adhesion molecule) and Sidekick (Sdk) cell adhesion molecules for potentially playing crucial roles in this system. In situ hybridisation in the embryonic mouse visual system showed Dscam and Sdk-1 expression in the RGC layer of the retina, along the optic pathway and in the visual targets. Sdk-2 was detected in the glia of the optic nerve and optic chiasm, marking the pathway that RGC axons follow, but not in RGCs. No DscamL1 was detected in RGCs or the optic pathway at the stages investigated and it was discounted from future analysis. In vitro, DSCAM promoted RGC axon outgrowth, whereas SDK 1 was inhibitory. SDK 2 had no effect on RGC axon outgrowth, suggesting it does not play a direct role in their pathfinding. Repeating this assay using retinal explants from the Dscamdel17 mouse mutant, showed that DSCAM enhanced retinal axon outgrowth, at least in part, through homophilic interactions. Analysis of visual system development in Dscam mutants showed DSCAM involvement in RGC axon fasciculation and in enhancing their growth, particularly within the ipsilateral optic tract. Retinal cell counts revealed that DSCAM played diverse roles in controlling cell number. Pre- and postnatal retinas lacking DSCAM contained more RGCs and mitotic cells. Postnatally, Dscam-/- retinas also show decreased cell death. In many cases, defect severity was dose-dependent, with an intermediate phenotype in the heterozygous mice, implicating DSCAM in the neurological defects of Downs’ Syndrome patients.