Enlightening axonal activity: Optical approaches to identify ion channels and their function

Abstract

In this thesis we asked the questions how ion signaling and the periodic organization at the AIS shape the generation of action potentials. In order to answer these questions, we need to bridge functional investigations with high resolution structural reconstructions. In Chapter 2, we used optical calcium recordings and identified three calcium entry pathways in the AIS. As expected, calcium is released from internal stores and enters through calcium channels, however, surprisingly, we also observed calcium entering through sodium channels. We estimated that the conductivity ratio of sodium channels for calcium is small, but because they are present at a high density at the AIS, they do form a major and rapid source of calcium. In Chapter 3, we investigated whether the calcium-dependent BK channel was a downstream target for calcium in the AIS. We implemented a novel technique to use light patterning of a fluorescent voltage reporter to obtain highly accurate measures of the action potential shape in the axon. BK channels were indeed activated during the action potential at the AIS, forming a link between calcium entry and action potential repolarization. Together, the complex of calcium and BK channels mediated high-frequency burst firing, an important feature of the cell type that we studied. In Chapter 4, we developed a novel optical method to perform high resolution microscopy deep inside tissue, where the neurons are in an intact three-dimensional context. Because biological tissue is not transparent, light traveling through tissue suffers from distortions, which makes microscopy at depth problematic. To overcome this obstacle, we used a deformable mirror to counteract the light distortions and enable high resolution microscopy inside biological tissue. We used this method to perform both live experiments and high-resolution microscopy from the same neuron, demonstrating that this method can bridge the structure-function relationship in neurons. Together, the experiments in this thesis shed light on the biophysical properties of axonal ion fluxes and how they are tuned to regulate proper neuronal excitability. The work presented in this thesis shows that optical approaches provide valuable tools in neuroscientific research and open novel avenues for future investigation of the biophysical properties of the neuronal membrane.