Clark, AJ; (2014) The Use of Microfluidic Chambers to Study Action Potential Propagation and Stimulus Transduction in Sensory Neurons in Vitro. Doctoral thesis (PhD), UCL (University College London).

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Abstract

Primary afferent sensory neurons can be incredibly long single cellular structures, often traversing distances of over one metre in the human. Cutaneous sensory stimuli are transduced in the periphery by specialised end-organs or free nerve endings which enable the coding of the stimulus into electrical action potentials that propagate towards the central nervous system. Despite significant advances in our knowledge of sensory neuron physiology and ion channel expression, many commonly used techniques fail to accurately model the primary afferent neuron in its entirety. In vitro experiments often focus on the cell somata and neglect the fundamental processes of peripheral stimulus transduction and action potential propagation. Despite this, these experiments are frequently used as a model for cellular investigations of the receptive terminals. Crucially, somal responses may not represent the functional expression of ion channels in the axon and end terminals. The aim of this thesis was to develop a system using compartmentalised culture chambers and ratiometric calcium imaging to directly and accurately compare the sensitivity and functional protein expression of isolated neuronal regions in vitro. Using this preparation I demonstrate that the nerve terminals of cultured DRG neurons can be depolarised to induce action potential propagation, which has both a TTX-resistant and TTX-sensitive component. Furthermore, I show that there is a differential regulation of proton sensitivity between the sensory terminals and somata in cultured sensory neurons. I also go on to show that capsaicin sensitivity is highly dependent on embryonic dissection age. This novel approach enables a comprehensive method to study the excitability characteristics and regional sensitivity differences of cultured sensory neurons on a single cell level. Examination of the sensory terminals is crucial to further understand the properties and diversity of DRG sensory neurons.

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