Author ORCID Identifier

0000-0002-5431-0309

Date of Award

2024

Document Type

Thesis (Ph.D.)

Department or Program

Integrative Neuroscience

First Advisor

Michael Hoppa

Second Advisor

Robert Hill

Third Advisor

Robert Maue

Abstract

Synaptic connections throughout the nervous system exhibit two forms of neurotransmitter release caused by synaptic vesicle fusion, evoked and spontaneous release. Despite being discovered 70 years ago, the mechanisms governing spontaneous release have remained elusive. Our understanding of spontaneous release lacks mechanistic details including discrepancies in the sources of synaptic vesicles supporting it, the source(s) of calcium supporting it, and most importantly an unclear relationship (cooperative or antagonistic) with evoked release. These gaps exist due to a lack of experimental methods specific to the study of spontaneous release at individual synapses.

This thesis addresses these gaps in two ways. Firstly, we address the use of fluorescent indicators for the study of spontaneous release. This includes characterizing numerous fluorescent indicators for measuring calcium and release of glutamate in hippocampal neuron cultures. We hypothesize that small sub-action potential level calcium transients are likely responsible for spontaneous vesicle release. However, we show that modern calcium indicators lack the sensitivity to detect these small changes in intracellular calcium. We also test a number of optimized glutamate indicators and collaborate with a team to develop and validate one of these indicators, iGluSnFR3. We demonstrate that iGluSnFR3 has the sensitivity and photostability to detect single vesicle release events at individual presynaptic hippocampal boutons in cultured neurons, without saturation, allowing quantitative measurement of critical aspects of evoked transmission.

The second way we address gaps in the study of spontaneous release is by using the newly optimized iGluSnFR3 indicator to correlate the size of the readily releasable pool (RRP) of vesicles to the frequency of spontaneous release at individual presynaptic boutons. By utilizing this new technique, we show a cooperative relationship between spontaneous and evoked release at the level of individual synapses. By and large, synapses do not specialize in one form of release but instead, those exhibiting high levels of spontaneous release also have higher evoked release amplitude, which we attribute to higher RRP sizes at these boutons. We go on to show that acutely increasing the size of the RRP through different but complementary pathways results in a near doubling of spontaneous release frequency, suggesting spontaneous release can be acutely modulated through dynamic changes to the RRP. Finally, we show that spontaneous release can be independently regulated as form of neurotransmission outside of the RRP, likely through voltage-gated calcium channels.

Original Citation

Aggarwal A et al. (2023) Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission. Nat Methods:1–10.

Ralowicz A.J. et al (2024) Frequency of spontaneous neurotransmission at individual boutons corresponds to the size of the readily releasable pool of vesicles. Journal of Neuroscience 21 February 2024, e1253232024

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