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In this project we used a combination of electrophysiology and fluorescent imaging to monitor synaptic transmission and calcium signalling in synaptic terminals. The study as a whole intended to examine how presynaptic calcium contributes to normal synaptic transmission within different hippocampal neuronal pathways. To this end, we used a transgenic mouse strain known as SyG37 that stably expresses a calcium sensor, SyGCaMP2-mCherry that is expressed in subsets of CNS neurones under the control of the Thy1 promoter. Our findings indicate that this new ratiometric sensor, in the SyG37 mouse strain, provides an excellent tool for detecting neural activity in acute brain slices. First, we showed that evoked calcium transients can be detected in acute brain slices prepared from SyG37 mice where electrical activation of Schaffer collaterals or mossy fibres elicited large calcium transients in area CA1 and CA3, respectively. Using immunohistochemical techniques, SyGCaMP2-mCherry co-localised with presynaptic proteins such as Bassoon, VGLUT1 and VGAT, confirming that it is expressed presynaptically in both excitatory and inhibitory terminals. Blocking fast glutamatergic and GABA/Glycinerergic transmission reduced the size of calcium transients in CA1 and CA3 by only 25 and 20% respectively indicating that the majority of the signals originated from first order presynaptic terminals. Pharmacologically, manipulating the adenosine receptor signalling pathway showed that the actions of adenosine, via the A1 receptor subtype, were different in the CA3 region compared to those in CA1. Forskolin also caused a small, concentration dependent effect on SyGCaMP2 fluorescence in response to electrical stimulation within both CA1 and CA3 regions with pronounced effects on field potential recordings. Together, with this SyG37 strain of transgenic mouse, it is possible to detect neuronal activity with fast temporal and high spatial resolution without the need for pre-incubation with organic calcium dyes or invasive viral transduction procedures.

Monitoring Ca²⁺ in intracellular compartments remains an uphill task. Fluorescent, organic calcium dyes and genetically encoded calcium indicators (GECIs) have been widely used to increase our understanding of neural activity. Notable among the GECIs is the GCaMP family. The first versions of this family showed potential but were relatively dim and had poor signal to noise ratios. To overcome these weaknesses, our laboratory developed three new ratiometric versions of the SyGCaMP2 family called SyGCaMP2-mCherry, SyGCaMP2-mCherry⁺ and GCaMP2-mCherry. The purpose of the work presented here was to establish whether these sensors were functional and to then use them to examine calcium signalling within the presynaptic terminals of cultured hippocampal and cerebral cortical neurones. The sensors were expressed in HEK 293T cells and their sensitivities to changes in intracellular free Ca²⁺concentration ascertained. The dissociation constants calculated for SyGCaMP2-mCherry⁺ and SyGCaMP2-mCherry were 140 ± 3.1 nM and 149 ± 3.3 nM respectively, but GCaMP2-mCherry was not accurately measured. Sensors were expressed in cortical and hippocampal neurones using lipofection based transfection methods and the expression patterns of each recorded. Both SyGCaMP2-mCherry and SyGCaMP2-mCherry⁺ were expressed in puncta that co-localised with presynaptic markers bassoon and VGlut1. Neurones were activated using field stimulation and the responses to different intensities and patterns of stimulation evaluated. Using the Thy1.2 promotor for neuronal expression, two transgenic mice (SyG 14 and SyG 37) were engineered in our lab that expressed SyGCaMP2-mCherry and responses to electrical stimulation were characterised in hippocampal brain slices. The roles of intracellular Ca²⁺stores in shaping the presynaptic Ca²⁺dynamics were examined and results demonstrated that inhibiting the sarcoendoplasmic reticulum calcium transport ATPase (SERCA pump) with Cyclopiazonic acid (CPA) and thapsigargin led to an enhancement in synaptic strength. In addition, activating ryanodine receptors (RYRs) with caffeine and low concentrations of ryanodine altered presynaptic Ca²⁺ dynamics.

Serotonin (5HT) and nitric oxide (NO) are important signalling molecules with roles in behavioural control and various psychological conditions. Various lines of evidence suggest interactions between 5HT and NO signalling, however little is known about their interaction in modulating intrinsic properties of cortical neurons and the effects on the neuronal network activity in the cortex. This project studied the effects of 5HT and NO on cultured neurons isolated from Wistar rat cortices. 5HT and NO produced diverse responses in cortical neurons, reflecting the heterogeneity of cortical neuron types. Neither 5HT nor NO appeared to significantly modulate glutamate induced depolarisations. However, they affected the number of APs elicited by the glutamate response, hinting at the possible modulation of intrinsic membrane properties. Subsequently, k-Means clustering based on 10 electrophysiological parameters was used to divide cultured cortical neurons into 10 different cell types with consistent firing properties. 5HT and NO had diverse, cell-type specific effects on neuronal excitability and cellular parameters in these cell types. Furthermore, 5HT and NO showed complex interactions between each other, suggesting modulatory cross-talk. Using ODQ to block soluble guanylate cyclase activity suggests that NO effects are mediated not only by sGC/cGMP signalling, but also S-nitrosylation. Comparing results obtained in cell culture to data from acute cortical slices showed similarities in basic cellular properties. However, 5HT and NO effects on pyramidal cell properties differed markedly between slices and cultures. At the network level, 5HT increased the spontaneous EPSC frequency in pyramidal neurons through 5HT2 receptors, whereas NO had no effect. Interestingly, NO was able to reduce the 5HT induced increase in EPSC frequency when inhibitory synaptic interactions were blocked, possibly by modulating 5HT1 receptor function. The results obtained demonstrate the interaction between 5HT and NO in modulating both intrinsic membrane properties and neocortical network activity.

The demarcation membrane system (DMS) is an extensive plasma membrane invagination system of the megakaryocyte (MK) that provides a membrane reserve for platelet generation. The properties of the DMS are poorly understood, particularly in living MKs. In this study, advanced electron microscopy, live cell confocal imaging and pharmacological tools were used to study the DMS and its connections with the extracellular environment. Confocal imaging of membrane-impermeant extracellular fluorescent indicators (HPTS, FITC-dextrans between 4 and 2000 kDa, and quantum dots) provided evidence for a discrete molecular cut-off (≈110kDa, estimated to be ≈11.0 nm by dynamic light scatter (DLS) measurements) for access to the DMS, which will prevent entry of large adhesion molecules (e.g. fibrinogen). Using extra-high resolution scanning electron microscopy (SEM) of fixed MKs, membrane invagination pores (MIPs) were observed on the MK surface that were variable in size but generally larger than predicted from live cell imaging experiments. A neck constriction was observed beneath the surface opening of the DMS using Gatan 3-view serial block face EM and may explain the rejection of molecules smaller than the surface pore. After inhibition of Cdc42, the DMS allowed entry of all indicators tested (up to 2000 kDa = reported to be ≈ 54.0 nm diameter). Using membrane-impermeant fluorescent indicators, electrophysiological capacitance measurements and transmission EM, multiple cationic amphiphilic drugs (CADs) were shown to cause complete surface detachment of the DMS. These compounds included the calmodulin inhibitor W-7, the phospholipase-C inhibitor U-73122 and anti-psychotic phenothiazines (trifluoperazine and chlorpromazine). CADs also caused loss of T tubules in cardiac ventricular myocytes and the open canalicular system of platelets. A possible explanation for this action is the ability of CADs to interfere with the interaction between PIP2 and binding of BAR domain containing proteins or the cytoskeleton. This work provides new insights into plasma membrane invagination systems.