Development of a novel optical probe to study functional heterogeneity of individual neurotransmitter release sites at the neuromuscular junction

About This Grant

Project Summary: Studies of presynaptic physiology at the neuromuscular junction (NMJ) have traditionally relied on the assumption that the probability of release is homogeneous across the hundreds of release sites or active zones at these synapses. However, a growing collection of evidence suggests that the probability of release varies widely across active zones at the NMJ. There is currently no method to quantify the spatial distribution of active transmitter release sites with single vesicle resolution at mammalian NMJs. Therefore, our main objective is to develop and use a new approach to characterize the probability of neurotransmitter release at individual active zones of mouse NMJs by improving upon previous attempts to visualize neurotransmitter release. We propose to investigate heterogeneity in probability of release across the hundreds of mouse active zones at the NMJ in the context of normal physiological and diseased conditions. We aim to demonstrate this using a novel optical probe to resolve neurotransmitter release at the level of a single quantal release event. For this purpose, we developed an optical probe by conjugating an intensity-based acetylcholine sensing fluorescent reporter, iAChSnFR to an scFv antibody fragment directed against nicotinic acetylcholine receptors (mAb35) that does not interfere with the normal functioning of the receptor channel. Frist, we will characterize the probability of transmitter release in healthy mouse NMJs. We will visualize synaptic vesicle release events using our novel acetylcholine receptor-targeted acetylcholine sensor (iAChSnFR-mAb35) in the mouse epitrochleoanconeus muscle and then calculate the probability of neurotransmitter release from each live iAChSnFR-mAb35 transient recorded during synaptic activity by mapping these events onto the localization of presynaptic active zones. Second, we will repeat this approach in the amyotrophic lateral sclerosis (ALS) model mouse NMJ evaluating the impact of disease progression. We hypothesize that the regulation of transmitter release within active zones may be different when comparing healthy and ALS NMJs. The proposed work promises to provide an important new tool for studying the probability of neurotransmitter release and resolving uncertainties in the heterogeneity of individual transmitter release-site function by correlating active zone structure and function under normal physiological conditions and in disease. Such an investigation promises to transform the ways in which we study and understand presynaptic physiology and modulation of neurotransmitter release in healthy and disease states.