THE DETECTION AND ANALYSIS OF ‘PRESYNAPTIC’ MECHANISMS MODULATING EXOCYTOTIC NEUROTRANSMITTER RELEASE EVENTS FROM SINGLE PC12 CELLS USING AMPEROMETRY AND TRANSMISSION ELECTRON MICROSCOPY

Open Access

Author:

Sombers, Leslie A.

Graduate Program:

Chemistry

Degree:

Doctor of Philosophy

Document Type:

Dissertation

Date of Defense:

March 15, 2004

Committee Members:

• Andrew Ewing, Committee Chair/Co-Chair
• Anne M Andrews, Committee Member
• Christine Dolan Keating, Committee Member
• Richard W Ordway, Committee Member

Keywords:

• Fusion pore
• exocytosis
• amperometry
• transmission electron microscopy
• PC12 cells
• vesicle volume
• foot

Abstract:

Understanding vesicular exocytosis is fundamental to developing insight into chemistry, biology and physics of neurotransmission. Through the direct ‘presynaptic’ observation of quantal release using amperometry it becomes strikingly evident that the exocytotic release event is plastic and regulated by multiple mechanisms. Electrochemistry is particularly useful for these types of studies as it provides highly sensitive qualitative and quantitative information for electroactive neurochemicals that are easily oxidized or reduced. The overall goal of this thesis is to use amperometry, in conjunction with electron microscopy, for the study of exocytosis events. By combining these approaches it is possible to study exocytotic mechanisms in more detail and thus to better understand specific regulatory aspects of communication between single cells. Exocytosis has been studied by a variety of methods; however, when living cells are used it is difficult to discriminate between the molecular effects of membrane proteins and the mechanics of lipid-membrane driven processes. Thus, this thesis describes the use of a novel liposome-lipid nanotube network as an artificial cell model that undergoes the later stages of exocytosis. This model shows that membrane mechanics, even without protein intervention, are sufficient to drive the expansion of the fusion pore to the final stage of exocytosis and can affect the rate of transmitter release through the fusion pore. The bulk of this thesis describes the analysis of exocytosis at a single PC12 cells. Many spikes in amperometric records of exocytosis events from many different cell types initially exhibit a pre-spike feature, or foot, which represents a steady-state flux of neurotransmitter through a stable fusion pore spanning both the vesicle and plasma membranes and connecting the vesicle lumen to the extracellular fluid. It has been well established that the volume of secretory vesicles can be modulated. Results presented in this thesis are the first evidence indicating that vesicular volume prior to secretion is strongly correlated with the characteristics of amperometric foot events. Amperometry and transmission electron microscopy have been used to determine that as vesicle size is decreased the frequency with which foot events are observed increases, the amount and duration of neurotransmitter released in the foot portion of the event decreases, and vesicles release a greater percentage of their total contents in the foot portion of the event. These previously unidentified correlations provide new insight into how vesicle volume can modulate the activity of the exocytotic fusion pore. Amperometric data have also been collected from PC12 cells in high osmolarity saline solutions, and these data are used to further clarify the biophysical characteristics of the fusion pore. When exocytosis is measured under high osmolarity conditions, dissociation of the dense core vesicle matrix is largely inhibited and thus membrane properties and release dynamics are changed. As a result, the shape of amperometric spikes is drastically altered with the foot portion of the spike exhibiting the most significant alterations from normal spikes. When release is elicited in high osmolarity saline, PC12 cell dense core vesicles are delayed after opening of the fusion pore and before proceeding to full vesicular fusion. Large dense core vesicles in PC12 cells are commonly loaded with dopamine by treatment with L-3,4-dihydroxyphenylalanine (L-DOPA). Indeed, many of the studies presented in this thesis utilize L-DOPA as a means of increasing vesicular volume. The results presented indicate that the majority of the dopamine loaded into these vesicles is preferentially compartmentalized into the halo portion of the vesicle. Amperometric records of release from cells stimulated in isotonic and hypertonic extracellular conditions have been compared. This is useful because the latter condition has been shown to cause inhibition of dense core dissociation in neuroendocrine cells, thus allowing for release specifically from the vesicular halo. In combination with this, transmission electron microscopy has been used to determine the morphological characteristics of dense core vesicles before and after treatment with L-DOPA in saline solutions of varied osmolarity. The results provide a more complete understanding of the complex interaction of molecules within dense core vesicles, suggesting that newly loaded dopamine is located in the halo and that the function of the dense core involves more than simple storage of neurotransmitter and associated molecules. Transmission electron micrographs of PC12 cells presented in this thesis indicate that some dense core vesicles contain multiple cores. These multi-cored vesicles, which are significantly increased in volume as compared to vesicles with a single dense core, are most frequently observed in PC12 cells bathed in high osmolarity, L-DOPA containing saline solutions. The data suggest that multiple-cored vesicles are formed when a population of single-cored vesicles homotypically fuses prior to regulated exocytosis. As these larger, multi-cored vesicles contain a greater amount of neurotransmitter, they should release more neurotransmitter and should give rise to longer and larger pre-spike amperometric foot signals, thus leading to another mechanism by which cells can modulate communication events.