Student Theses and Dissertations

Date of Award

2017

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Bargmann Laboratory

Abstract

Chemical synapses are complex structures that have a diversity of specific activities that shape nervous system computation. To understand how this diversity contributes to specific circuit functions, we sought to characterize synaptic release in the stereotyped and defined neural circuitry of C. elegans. Here we use pHluorin imaging (Vglut-­pH) to monitor presynaptic glutamate release from single chemosensory neurons in intact animals and characterize the dynamics of endo-­ and exocytosis from two types of glutamatergic neurons, AWCON and ASH. In Chapter 1, we describe the optimization of Vglut-­pH, we introduce a reagent for measuring synaptic calcium influx by tethering GCaMP to synaptic vesicles, and we provide our initial characterization of glutamate release in AWCON and ASH. Our results indicate that AWCON and ASH have distinct exocytosis dynamics and that AWCON exhibits synaptic release properties similar to vertebrate photoreceptors and retinal bipolar neurons, aligning with previous results from functional calcium imaging, gene expression, and circuitry. In Chapter 2, we characterize the dynamics of endocytosis in AWCON and ASH. We find that synaptic vesicle endocytosis in these neurons have kinetic features and timescales similar to that of mammalian neurons. We show that endocytosis appears to be homeostatically regulated by previous neuronal activity and is composed of at least two kinetically distinct modes, fast and slow. We show that fast retrieval is dependent on the clathrin adaptor protein AP180/CALM, suggesting that clathrin-­mediated endocytosis is important for synaptic vesicle retrieval in these sensory neurons. In Chapter 3, we compare and contrast the dependences of AWCON and ASH Vglut-­pH responses on the core synaptic vesicle release machinery and its regulators: syntaxin, synaptobrevin, SNAP-­25, unc-­13, unc-­18, RIM, complexin, and tomosyn. We find that Vglut-­pH responses are highly dependent on the core components of the SNARE complex and its regulators, but we detected significant differences in the residual responses in these mutants that suggest AWCON and ASH synapses are distinct from each other and from those of the neuromuscular junction. We find that complexin appears to act as an inhibitor of SV release in AWCON and ASH, and we find an unexpected role for tomosyn in regulating calcium influx. In Chapter 4, we describe activity-­dependent cytoplasmic pH changes in AWCON and ASH, and we conduct a series of experiments to show that these pH changes do not interfere with the measurement or interpretation Vglut-­pH signals. Our results indicate that these activity-­dependent pH changes are consistent with a depolarization-­generated acidosis and are correlated with calcium influx. We show that these pH changes in response to stimulation are not dependent on unc-­13 or unc-­18 and are thus are largely independent of synaptic release. Finally, we show that in contrast to intracellular pH, extracellular pH changes are not detected in response to sensory stimulation. In Chapter 5, we investigate the role of synaptotagmins in AWCON and ASH glutamate release using Vglut-­pH imaging. We find that AWCON basal release is highly dependent on snt-­1, whereas ASH exocytosis is intact in snt-­1 null mutants and slightly diminished in snt-­6 mutants. Our results indicate that AWC and ASH synapses have distinct requirements for snt-­1 and may use a combination of calcium sensors to mediate glutamate release. In Chapter 6, we use a combination of genetics, behavior, calcium imaging, and Vglut-­pH imaging to investigate how loss-­of-­function mutations in pkc-­1 (protein kinase C epsilon) modulate AWCON butanone olfactory preference. We find that pkc-­1 functions in AWCON downstream of presynaptic calcium influx to modulate eat-­4 dependent glutamate release and a second form of AWCON output that is important for specifying butanone olfactory preference. We identify the receptor-­type guanylate cyclase gcy-­28 and Gqα as additional important regulators of AWCON synaptic release, and identify unc-­31 (CAPS) as an additional genetic determinant of butanone olfactory preference. Finally, we suggest a model for a dual function Gqα/DAG/pkc-­1 signaling pathway that regulates synaptic vesicle release and butanone preference in AWCON. Our work in this thesis extends the characterization of C. elegans synapses from the neuromuscular junction to the presynaptic terminals of central synapses and supports a role for presynaptic diversity among distinct neuronal cell types in C. elegans. Our work emphasizes that presynaptic diversity and regulation of neurotransmitter release are important components to specifying circuit function and suggest that C. elegans will provide a deeper understanding of how presynaptic diversity, both in terms of molecular components and activity dynamics, contribute to nervous system function.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Available for download on Wednesday, August 01, 2018

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