Ionotropic and metabotropic signalling in neuronal development and differentiation

This thesis investigates ionotropic and metabotropic signalling mechanisms in developing neurons from human embryonic stem cell and primary sources. Focus is placed on the measurement of functional activity using primarily whole-cell patchclamp and Ca2+ imaging techniques These signalling mechanisms were investigated in undifferentiated human embryonic stem cells, hESC-derived neurons, fetal primary human neurons and neonatal primary mouse neurons. The results of this research are separated into three chapters. Preliminary work carried out on iPSC-derived neurons is also included as an indication of future direction Chapter 3: P2 Receptors in hESCs Purinergic signalling was shown to be active in undifferentiated hESC populations. Specifically, the activity of P2Y1 receptors was confirmed pharmacologically. This is a novel observation and indicates a mechanism for physiologically relevant signalling molecules to modify [Ca2+]i Chapter 4: Functional Characterisation of hESC-Derived and Primary Neurons Functional characteristics associated with neuronal development were measured in human embryonic stem cells during terminal neuronal differentiation in a chemically-defined medium. The presence and activity of voltage-gated Na+, K+ and Ca2+ channels were recorded, alongside data on neuronal excitability (Vm, iAP induction and threshold and spontaneous electrical activity). These data were also recorded in fetal hWGE- and neonatal mWGE-derived neurons for comparison. hESC-derived neurons were shown to be functionally more similar to fetal hWGEneurons suggesting an immature neuronal phenotype Chapter 5: GABAergic Signalling in hESC-Derived and Primary Neurons GABAergic signalling in hESC-, mWGE- and hWGE-derived neuronal populations was investigated. Focus was placed on hWGE-derived neurons and the developmental state of GABAergic responses. In fetal hWGE-derived neurons, a percentage of cells displayed an ‘inhibitory’ response to GABAAR activation. This is a novel observation with implications in human neuronal development. In vitro modulation of GABAergic signalling was also shown, providing potential tools for future research into this phenomenon Chapter 6: Future Developments and General Discussion iPSC-derived neuronal populations were shown to display basic neuronal functional properties. This work will form the basis of future studies on these cell