Spatiotemporal regulation of neurotransmission at single synapses

About This Grant

PROJECT SUMMARY The release of neurotransmitters at synapses occurs in three distinct modes: synchronous release, which is tightly coupled to an action potential (AP); asynchronous release, which lingers for hundreds of milliseconds after an AP; and spontaneous release, which happens independently of an AP. While our labs and others have begun uncovering the molecular machinery behind asynchronous release, major gaps remain – especially at the quantal level. This is largely because most of what we know about neurotransmitter release comes from electrophysiological studies, which lack direct spatial resolution. To address this problem, I will leverage the power of iGluSnFR3, a third-generation intensity-based Glutamate-Sensing Fluorescent Reporter, to directly visualize asynchronous release at the single-synapse level and dissect its molecular and physiological drivers. Aim 1 will determine if distinct molecular drivers of asynchronous release generate unique quantal and spatial phenotypes at the single synapse. First, I will further optimize iGluSnFR3 for reliable detection of quantal neurotransmitter release (subaim 1.1). My preliminary data demonstrates that iGluSnFR3 can detect quantal neurotransmission with single-synapse resolution. Subaim 1.2 will investigate how different extrinsic and physiological conditions can uniquely desynchronize neurotransmitter release. Although these conditions are often used interchangeably in the literature and assumed to have the same effect on facilitating asynchronous release, it remains unclear whether they influence release kinetics at the single-synapse level in the same way. My preliminary data shows that while these conditions increase the cumulative charge transfer of asynchronous release measured by electrophysiology, iGluSnFR3 imaging reveals striking differences at the single-synapse level. Finally, subaim 1.3 will investigate whether this desynchronization is organized within separate sub- domains of the same active zone. Aim 2 will test whether synaptotagmin-7 mediated asynchronous release regulates postsynaptic signaling (subaim 2.1) and plasticity thresholds (subaim 2.2). My preliminary data indicates that synaptotagmin-7 not only drives increased multivesicular release but also elevates postsynaptic calcium levels, leading to the activation of downstream target proteins. The overall hypothesis of my proposal is that asynchronous neurotransmission constitutes a distinct signaling pathway, separate from other modes of release, with unique regulatory mechanisms and functional consequences. Therefore, the completion of this project will further our understanding of the molecular mechanisms shaping neurotransmission and synaptic communication. Additionally, this F31 proposal, supported by a highly individualized mentoring plan and team, will help prepare me for my next career stage as I apply to psychiatry research-track residency programs, and, ultimately, to become an independently funded physician-scientist.