Ginebaugh, Scott Patrick
(2021)
Short duration presynaptic action potentials shape calcium dynamics and transmitter release at the neuromuscular junction in healthy and diseased states.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
The action potential (AP) waveform controls the opening of voltage-gated calcium channels (VGCCs) at presynaptic nerve terminals. The calcium ion flux through these VGCCs acts as a second messenger, triggering the release of neurotransmitter. The frog and mouse neuromuscular junctions (NMJs) have long been model synapses for the study of neurotransmission, but the presynaptic AP waveforms from these NMJs have never been recorded because the nerve terminals are too small impale with an electrode for electrophysiological recordings. Neurotransmission from nerve terminals occurs at highly organized structures called active zones (AZs), and the relationships between the AP, VGCCs, and neurotransmission at AZs are poorly understood. Understanding these relationships is important for the study of Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disorder in which neurotransmitter release from the NMJ is decreased, leading to severe muscle weakness. This reduced neurotransmission is thought to be caused by an antibody-mediated removal of presynaptic VGCCs and disruption of AZ structure. Furthermore, 3,4-diaminopyridine (3,4-DAP; the FDA-approved treatment for LEMS) was thought to indirectly increase calcium flux into the AZs by broadening the presynaptic AP, but this mechanism has come under scrutiny. Here, we use voltage imaging to optically record AP waveforms from frog and mouse motoneuron terminals. We find that the AP waveforms from these terminals are very brief in duration. We hypothesize the brief duration of these APs helps prevent a depletion of docked synaptic vesicles in AZs by limiting calcium flux during APs, and is thus an important mechanism by which healthy NMJs maintain reliable neurotransmission during repeated stimulation. We also show that clinically relevant concentrations of 3,4-DAP increase calcium flux by broadening the AP. Next, we use our recorded AP waveforms to constrain computational models of mammalian AZs, and utilize these models to investigate the effects of LEMS on the AZ. We demonstrate that the disruption of AZ structure plays an essential role in LEMS pathology. Finally, we show that disrupting the calcium-sensing proteins in the AZ could result in LEMS pathology and hypothesize that anti-VGCC antibodies may not be solely responsible for LEMS in many LEMS patients.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
31 March 2021 |
| Date Type: |
Publication |
| Defense Date: |
27 January 2021 |
| Approval Date: |
31 March 2021 |
| Submission Date: |
3 February 2021 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
199 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Integrative Systems Biology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
neuromuscular junction, voltage imaging, Lambert-Eaton myasthenic syndrome, MCell, calcium dynamics, active zone, action potential, synaptic transmission |
| Date Deposited: |
31 Mar 2021 15:43 |
| Last Modified: |
31 Mar 2021 15:43 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/40222 |
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