Vesicular Loading and Synaptic Release at Central Cholinergic Synapses

Abstract

Neurotransmitter release from presynaptic terminals can be regulated by altering transmitter load per synaptic vesicle (SV) and/or through the probability of vesicle release. The vesicular acetylcholine transporter (VAChT) loads acetylcholine (ACh) into SVs at cholinergic synapses. This project presents three major aims; to investigate the physiological implications of (i) VAChT activity modulation in central cholinergic physiology (ii) insecticide resistance through VAChT mutagenesis of known resistance mutations and (iii) SV loading through structural manipulation of the VAChT C-terminal polyQ region. Chapter 2 of this thesis investigates how pharmacological blockade (5Cl-CASPP exposure) and transgenic up-regulation of VAChT affects SV content and release frequency at central synapses in Drosophila melanogaster and provides strong evidence that vesicle loading follows a set point model. Chapter 3 examines cholinergic dysfunction in two 5Cl-CASPP resistant genetic backgrounds (VAChTY49N and VAChT up-regulation). Both resistance modalities increase spontaneous release frequency suggesting release frequency appears deterministic of CASPP activity. However, the VAChTY49N mutation additionally disrupts action potential-evoked cholinergic release and fictive locomotor patterning through depletion of releasable synaptic vesicles indicative of a functional trade-off. Chapter 4 investigates loading disruption through direct mutagenesis of the VAChT polyQ region. Truncation of the polyQ region, by one glutamine residue, results in increased SV loading kinetics whereas polyQ extension is sufficient to reduce loading. Deletion of the polyQ region has no obvious effect on spontaneous release, but evoked synaptic currents show increased duration. Thus the polyQ region of the insect VAChT is required for correct vesicle transmitter loading.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Hugh Piggins (Supervisor) & Richard Baines (Supervisor)