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Title: Functional properties of action potential propagation in sensory neuron cultures : an in vitro microfluidic investigation
Author: Morice, O. S.
ISNI:       0000 0004 8498 6400
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2017
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The discovery of the molecular identity of voltage-gated ion channels that underlie the initiation and propagation of action potentials has brought about formidable momentum in the elucidation of their contribution to electrogenesis and impulse propagation. Despite the characterisation of their unique biophysical properties, and the unravelling of their exquisite expression patterns in sensory classes, our understanding of their functional significance to the conductile function remains largely speculative. The aim of this project was to exploit the microfluidic technology, a newly-developed platform that allows the segregation of neuronal regions, to pharmacologically probe the developmental emergence of sodium channel isoforms, their regional patterns of expression, and their respective role to the conductile function. To this end embryonic and neonatal dorsal root ganglia neurons were seeded in microfluidic chambers and neuritic extensions directed chemotactically with a nerve growth factor gradient across the compartments. Ratiometric calcium imaging, using Fura-2, was used to visualise the somal response after depolarisation of the terminals with high KCl concentrations. Pharmacological agents were incubated in the different compartments. Following calcium imaging, neurons were identified by somal immunolabelling. In the first instance, I established and optimised a protocol to culture neonatal neurons in the microfluidic system. The majority of neurons extended neurites through the two arrays of microgrooves after 8 days in vitro, and were functionally responsive after such an incubatory period. In this system, neurons were repeatedly depolarised upon consecutive excitations on the terminal arbor, with the least tachyphylaxis observed with 100 mM KCl applications with a 5 minutes inter-stimuli recovery period. The propagation of action potentials from the terminals to the cell bodies relied on the activation of voltage-gated sodium channels, whilst a minority necessitated that of voltage-gated calcium channels. Functional expression of TRPV1 on the somal membrane increased markedly during development, an increase that did not correlate 4 with the gradual upregulation of the TTX-resistant properties of action potential propagation. Both NGF and GDNF maintained the functional expression of TTXresistant channels, whilst retinoic acid induced a further upregulation in a timedependent manner. The application of VGSC subtype-specific blockers along the axonal compartment revealed the necessity of the activation and opening of sodium channel isoforms to the propagation of action potentials in the largely-nociceptive population. The ICA- 121431 compound, a selective blocker of Nav1.1, Nav1.2, and Nav1.3, did not impede the conductile function, with the same lack of effect observed with the selective Nav1.5 blocker Jingzhaotoxin-III, indicating the functional redundancy of these currents to impulse propagation. On the other hand, the highly selective Nav1.7 blocker ProTx-II peptide blocked a minority of neurons with 10 - 100 nM application, concentrations affecting mainly Nav1.7-mediated currents, whilst 1 μM A-803467 blocked a majority of neonatal neurons in both N52-positive and negative neurons, a concentration deemed selective for the Nav1.8 isoform. This indicates that Nav1.7-selective blockade may not effectively impede the conductile function of nociceptive axons, whilst Nav1.8 appears to play a significant role in conduction of nociceptors in vitro. The application of ascending concentrations of lidocaine in the microfluidic comparments demonstrated the fine-tuning of sodium channel expression along the neuritic length, with the intermediate segment displaying an exceedingly high safety factor of transmission. Interestingly this region also displayed a high proportion of TTX-resistant conductance, relative to the more proximal and distal segments, further validating the fine regulation of sodium channel expression along the axolemma. The addition of GDNF in the cultures sustained the non-peptidergic population, increasing the proportion of neurons that displayed TTX-resistant properties of action potential propagation, whilst a further incubatory period did not alter the conductile properties, demonstrating that the expression of sodium channels is fully mature after 8 days in vitro in this system. This work provides the first systematic pharmacological investigation of the role of sodium channel isoforms to the conductile function in a native neuronal background. 5 It also sheds light on the functional regulation of TTX-sensitive and TTX-resistant conductance from a regional and developmental perspective. Taken together these findings may have important implications for the development of next-generation analgesics.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available