Despite its widespread use, a striking lack of knowledge exists regarding the mechanism of action of transcranial magnetic stimulation (TMS). This thesis describes the physiological characterisation of repetitive TMS (rTMS) to the motor system by means of functional magnetic resonance imaging (fMRI). A detailed analysis of imaging artefacts arising from the simultaneous application of TMS-fMRI was conducted and subsequently, strategies were presented for unperturbed TMS-fMRI. Physiological responses during subthreshold high-frequency rTMS of the primary sensorimotor cortex (Ml/Sl) were visualised within distinct cortical motor regions, comprising PMd, SMA, and contralateral Ml/Sl, while no significant responses were evidenced in the area of stimulation. Repetitive TMS during or before motor behaviour illustrated the context- dependence of rTMS-induced activity changes. The first demonstration of TMS-fMRI at 3 Tesla provided evidence that subthreshold rTMS can activate distinct networks including subcortical motor regions. The subthreshold nature of rTMS was confirmed by simultaneous electromyographic recordings from the target muscle. Stimulation of the dorsal premotor cortex provided evidence that rTMS- evoked local activity changes depend on the input function. The capability of TMS to target distinct networks in the human brain was confirmed. TMS targets a set of cortical and subcortical structures. Local responses may not invariably be elicited, indicating that low levels of synaptic activity, as occurring at low-intensity stimulation, do not necessarily evoke corresponding changes in cortical haemodynamics. It is concluded that combined TMS-fMRI offers a means to assess the mechanism of action of TMS at high spatial and temporal resolution.

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Haem oxygenase-1 (HO-1) is the inducible form of the enzyme responsible for the breakdown of haem, metabolising it to carbon monoxide (CO), bilirubin and iron. Protective properties have been attributed to HO-1 induction and haem breakdown products. Upregulation of HO-1 has been reported in many neurodegenerative disorders and it has been postulated that this may play an important role in the stress defences of the cell. Inducible nitric oxide (iNOS) activity, nitric oxide production and mitochondrial dysfunction are also reported in neurodegenerative disorders and it has been shown that inducers of iNOS also induce HO-1. Rat astrocytes have been used to examine possible interactions between the iNOS/HO systems by inducing iNOS activity with IFNy/LPS and HO-1 with hemin. It was confirmed that EFNy/LPS mediated NO production caused an upregulated HO-1 expression and an increase in HO activity. Further, it was shown that hemin treatment was able to prevent the previously reported NO mediated irreversible inhibiton of complex IV of the mitochondrial electron transport chain seen when astrocytes are treated with IFNy/LPS. Hemin plus IFNy/LPS treatment was accompanied by an increase in HO activity and an increase in glutathione (GSH) levels and it is postulated that together these confer protection. It may be concluded from this thesis that there are complex interactions between HO and NOS which could contribute to the ability of astrocytes to provide protection to neurones against oxidative and nitrosative stress. This may be relevant to mechanisms by which neurodegeneration occur.

Nitric oxide (NO) is a signalling molecule in the central nervous system and other tissues. NO synthesis and its immediate targets are well-characterised moleculaiiy, but little is known about how the NO signal is terminated. Recent work has suggested the existence of a biological sink for NO in dispersed brain tissue. This study aimed to discover the kinetic properties of NO inactivation in brain and to elucidate the mechanism(s) underlying this process. Measurements of cGMP accumulation in brain slices from cerebellum indicated that intact brain inactivates NO. When analysed using a model of NO diffusion and inactivation, the experimental data were consistent with the maximum rate of inactivation being fast (over 1 uM/s). It was inferred from the kinetics that biological inactivation of NO would predominantly affect NO signalling when several sources of NO are concurrently active. The mechanism of NO consumption initially studied in dispersed brain tissue has since been shown to be the reaction of NO with lipid peroxyls, a process that may have relevance to pathophysiology. Pharmacological and other tests showed that the breakdown of NO by cerebellar slices was, however, independent of this mechanism. Further manipulations also eliminated other potential routes of NO metabolism (reaction with red blood cells or superoxide, or autoxidation) as underlying causes. Lipid peroxide-independent NO inactivation was also found in acute and cultured cerebellar cells. The reaction exhibited marked oxygen- dependence. In dilute preparations, NO metabolism was largely lost on cell lysis, but was recoverable by addition of the electron donor NADPH. This activity resided in the membrane fraction following high speed centrifugation. Inactivation of NO in NADPH-treated lysates or membrane fractions, and in intact cells, was partially inhibited by cyanide. This evidence suggests the involvement of membrane-bound haem and flavoproteins. In concentrated tissue preparations and intact brain slices, however, an alternative process becomes dominant. Which of these mechanisms is most important physiologically requires further investigation. Their relative distributions with respect to the sites of NO release may be critical.

Ischaemia results from the cessation of blood flow to all or part of the brain, causing a rapid depletion of O2 and impairment of oxidative phosphorylation. Bioenergetic failure follows within minutes, which precipitates directly immediate neuronal death. Upon reperfusion, despite the return of energy substrates, further neuronal death can occur up to several days later, depending on the severity of the initial insult. Astrocytes have been shown to be considerably more resistant to ischaemia/reperfusion injury than neurones and may play either a neuroprotective role or indeed exacerbate the neuronal injury. We have explored the possible interactions between astrocytes and neurones following ischaemia using an in vitro model of ischaemia/reperfusion injury, as a controlled environment that lends itself more easily to manipulation of the numerous variables involved in such an insult. As such we have produced an oxygen glucose deprivation model. We have constructed a chamber in which O2 can be lowered to a concentration of 1 M. We have further developed a primary cortical neuronal culture that is 99% pure and which can survive to at least 10 days in vitro. We have additionally established a novel system for the co-culture of astrocytes and neurones in order to study the communication between these cells in a manner that can allow the complete separation of one cell-type from another. Astrocytes cultured alone do not exhibit signs of cell death during reperfusion of 24hrs duration following ischaemia of up to 2hrs whereas neuron cultures show profound cell death following an ischaemic period of only 15mins. We have co-cultured neurones, which have been subjected to a 15min ischemic insult, with either non-insulted astrocytes or astrocyte conditioned medium during the reperfusion stage. Results show that both astrocytes and astrocyte-conditioned medium enhance neuronal survival. We have finally investigated possible mechanisms to account for this.

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