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Title: Amperometric measurement and optogenetic manipulation of acetylcholine release
Author: Ruivo, Leonor M. Teles-Grilo
ISNI:       0000 0004 5917 227X
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2015
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Acetylcholine is one of many neurotransmitters engaged in modulating complex / behaviours. Cholinergic projections from the basal forebrain to the prefrontal cortex and the hippocampus are proposed to have an important role in regulating arousal and enhanced attention states, neuronal network activity, and synaptic plasticity during working memory 'functions. Supporting spatia~ working memory, the hippocampus is responsible for spatial navigation and encoding of new episodic memories, while the prefrontal cortex is engaged in top-down processing and handling of information from multiple sources to create cross-temporal and cross-modal associations that support goal-direct behaviour. With the aim to investigate the relationship between acetylcholine release and behavioural states switches, microelectrochemical biosensors were used to make continuous, sub-second resolution measurements of the spatiotemporal dynamics of acetylcholine release in the medial prefrontal cortex and dorsal hippocampus of mice. Acetylcholine release unfolded at two distinct time scales. Tonic acetylcholine release was coordinated in both brain regions, associated with levels of arousal and was dependent on the sequence of behavioural state transitions. Phasic acetylcholine release occurred during performance of a rewarded spatial working memory task. Phasic release events were detected preferentially in the maze reward delivery points regardless of trial outcome, pointing towards a role for acetylcholine in supporting cue-detection and placereward association. These data represent the first high-temporal resolution profile of acetylcholine release measured continuously across a number of different behavioural states, demonstrating the effectiveness of biosensors as a tool suitable to dissect in detail the role of acetylcholine in modulating brain states and behavioural output in vivo. In order to set up the techniques required to mimic in vivo acetylcholine release in hippocampal slices, optogenetic targeting of the septohippocampal cholinergic system was tested using conditional expression recombinant adeno and adeno-associated viral vectors, ChAT-Cre and channelrhodop$in knock-in mouse lines. Preliminary data on viral vector titration and channelrhodopsin expression time-course showed that transduction efficiency and specificity depended on the viral vector and channelrhodopsin mutant used. Widespread, targeted expression was observed following injections of a serotype-2 adeno-associated viral vector carrying the floxed, mCherry-tagged channelrhodopsin mutant H134R and in ChAT-Ai32 transgenic mice.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available