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Title: Developing optogenetics for use in vascular research
Author: Rorsman, Nils
ISNI:       0000 0004 7229 655X
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2017
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Optogenetics is a recently established experimental technique that involves the heterologous expression of light sensitive proteins (opsins) in mammalian cells to modulate cell function. One of the most commonly used opsin is the blue light gated depolarising channel, channelrhodopsin-2 (ChR2). Optogenetics involving ChR2 has revolutionised the field of neuroscience by enabling the definition of novel brain circuitries. Optogenetic control of the electromechanical coupling in vascular smooth muscle cells (SMCs) is now emerging as a powerful research tool with potential applications in drug discovery and therapeutics. However, the exact ionic mechanisms involved in this control remain unclear. The overall aim of this thesis was to establish ChR2 for use in vascular optogenetics. The main findings of this thesis are: 1) Blue light activation of ChR2 and the ChR2 variant ChR2(H134R) led to long-lasting and non-inactivating depolarising currents. 2) Transgenic mice expressing ChR2(H134R) selectively in SMCs were generated. Isolated SMCs obtained from these mice demonstrated blue light induced depolarising whole-cell currents. Fine control of artery tone was attained by varying the intensity of the blue light stimulus. This arterial response was sufficient to overcome the melanopsin-mediated light-depended arterial relaxation observed in the presence of contraction-eliciting agonists. 3) Pharmacological analyses revealed that Ca2+ entry through voltage-gated Ca2+ channels, and recruitment of plasmalemmal depolarising channels (TMEM16A and TRPM) and intracellular IP3 receptors were mandatory for the ChR2(H134R)-mediated arterial response to blue light at intensities <~0.1 mW/mm2. Light stimuli of greater power evoked a significant Ca2+ influx directly through ChR2(H134R) and produced dramatic intracellular alkalinisation of the SMCs. The light intensity range that enables optical control of arterial tone primarily through the recruitment of endogenous channels and without substantial alteration of intracellular pH, was identified. Within this range, mice expressing ChR2(H134R) in SMCs are a powerful experimental model for achieving accurate and tuneable optical voltage-clamp of SMCs and finely-graded control of arterial tone, offering new avenues to the discovery of vasorelaxing drugs. 4) The Cl--selective ChR2 mutant iChloC mediates depolarising currents while also preventing H+ or Ca2+ fluxes. This mutant was also found to have enhanced sensitivity to blue light. This new optogenetic tool could be a good candidate for an improved mouse model of vascular optogenetics. To conclude, the work presented in this thesis represents an in depth analysis of the use of ChR2(H134R) in vascular optogenetics. The cellular mechanism linking ChR2(H134R) opening with contraction of vascular SMCs was defined. Additionally, the limitations of the current optogenetic mouse model have been identified, and iChloC was shown to resolve these limitations. This study established vascular optogenetics as novel research tool vascular physiology and pharmacology.
Supervisor: Tammaro, Paolo Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available