Biological and Artificial Model Systems to Investigate Fundamental Properties and Structures Related to Exocytosis

Open Access
Adams, Kelly
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
November 05, 2009
Committee Members:
  • Dr, Andrew G Ewing, Dissertation Advisor
  • Andrew Ewing, Committee Chair
  • Anne M Andrews, Committee Member
  • Thomas E Mallouk, Committee Member
  • Peter J Butler, Committee Member
  • lipid nanotube
  • fusion pore
  • liposome artificial cell model
  • carbon fiber amperometry
  • exocytosis
  • microelectrode array
Neurotransmitter release via exocytosis, a fundamental process facilitating synaptic transmission in the brain, has been investigated by use of single or multiple carbon fiber microelectrodes, and by pairing with complementary optical microscopy techniques. The use of pheochromocytoma (PC12) and lipidic, artificial soybean liposome cell model systems are presented as means to examine the physical properties and structures influencing exocytotic release, specifically fusion pore size and stability, lipid membrane mechanics and composition, and pharmacological manipulation with a neuromodulator. First in Chapter 2, the effects of elevated osmolarity on neurotransmitter secretion from PC12 cells are presented. The number of pre-spike “feet” and the amount of transmitter released during these feet measured by carbon fiber amperometry were notably increased compared to control, a finding strongly suggesting that membrane mechanics play a modulatory role in presynaptic release by prolonging the lifetime of the fusion pore. Next in Chapter 3, the fabrication and characterization of a seven-barrel carbon fiber microelectrode array are presented. The tightly packed, individually addressed microelectrodes can be used to monitor exocytotic release from PC12 cells and events have been resolved at the sub-cellular level. In Chapter 4, the inhibitory effect of estrogen on depolarization-induced release of dopamine from PC12 cells is discussed. This effect is dose-dependent and involves N-type voltage-gated calcium channels, both results that have not been reported previously for this neuromodulator. Two concentrations of estrogen (10 nM and 50 µM) elicit release; however, intracellular calcium imaging reveals that calcium influx is significantly decreased for 10 nM E2, whereas it is unaffected for 50 µM E2. This suggests two probable mechanisms for estrogen: one involving voltage-gated calcium channels at low, physiological estrogen concentrations and another altering cell function at higher pharmacological estrogen concentrations. Such dissimilarity is important to consider when postulating a neuroprotective mechanism for estrogen. The thesis moves to the direct measurement of the dimensions of a lipid nanotube present in an artificial cell model for exocytosis in Chapters 5 and 6. A difference is shown to exist between situations when a micropipette is attached to the lipid nanotube which is then attached to a liposome (the tube-only case) versus when there is a vesicle is at the end of the micropipette (the two-vesicle configuration case). A method to electrochemically measure the inner diameter of the lipid nanotube is presented in Chapter 5 and these measurements as well as the differences between the tube-only and two-vesicle cases is with a theoretical model considering the different elastic free energy components of the entire system. Membrane composition and the effect of increased lipid concentration in the membrane are further explored in Chapter 6. Lipid composition has a direct effect on the measured size of the lipid nanotube, therefore implicating a more active role for phospholipids in influencing sizes of nanostructures in nature. Lastly in Chapter 7, a summary of this thesis as well as suggested future research directions aimed at furthering the understanding of neurotransmitter release are presented.