The hippocampus is a region of the brain recognised for its role in learning and memory. Underlying this function are changes in the strength of synaptic connections that arise due to previous experience. To enable molecular analysis of pathological mechanisms underlying disease processes, I set out to develop and characterise a tractable in vitro model of hippocampal neurons. This cell model was required to exhibit certain characteristics of neurons in vivo. In particular the neurons were synaptically connected, display action potentials and express functional metabotropic signalling systems. Ca2+ is a key neuronal signalling mediator controlling various functions such as membrane excitability, triggering the release of neurotransmitters, mediating activity-dependent changes in gene expression and modulating neuronal growth, differentiation and transition to apoptosis (Berridge 1998). To assess the functional properties of my hippocampal neuron culture, I therefore probed the development of Ca2+ signalling and the Ca2+ signalling proteome. By day 10 and 11 in vitro (DIV), hippocampal neurons exhibited spontaneous synchronous oscillations in their cytosolic Ca2+ that are a classical hallmark of a synaptically connected network. These spontaneous synchronous oscillations were inhibited by N-type but enhanced by L-type voltage-gated Ca2+ channel (VGCC) antagonists. At this stage of culture, the neurons also displayed elevated InsP3-mediated Ca2+ release from their endoplasmic reticulum Ca2+ stores and greater depolarisation-mediated Ca2+ signals. Quantitative analysis of confocal images of immunofluorescently labelled neurons at DIV 4, 8, 11 and 15 showed that neuronal arborisation and expression of the synaptic marker synapsin increased with age of the culture achieving a dense network by DIV 11. The expression levels of InsP3 5’-phosphatase, calreticulin, L- and N- type VGCCs remained constant up to and including DIV 8 and significantly increased thereafter coincident with the onset of Ca2+ oscillations. Expression of calnexin, PLCβ-1, InsP3 3’-kinase, InsP3R-1 and muscarinic GluR-5 proteins remained unchanged throughout maturation of the culture. Further investigations will establish the contribution of other components of the Ca2+ signalling toolkit in the generation of spontaneous Ca2+ oscillations. The role of these Ca2+ oscillations in neuronal maturation will subsequently be tested. Followed by the determination of the effect of prolonged enhancement or decrease in Ca2+ signalling during culture maturation and how it contributes to the onset and the quality of spontaneous synchronous oscillations.