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.
