UTILIZING IN VITRO CELLULAR SYSTEMS TO PROBE THE ROLE OF NEUROCHEMICALS ANALYTICALLY

Open Access
Author:
Arcibal, Imee G
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
September 15, 2009
Committee Members:
  • Andrew Ewing, Dissertation Advisor
  • Andrew Ewing, Committee Chair
  • Mary Elizabeth Williams, Committee Member
  • Anne M Andrews, Committee Member
  • Erwin A Vogler, Committee Member
  • Michael L Heien, Committee Member
Keywords:
  • Biogenic amines
  • exocytosis
  • PC12 cells
Abstract:
The dynamic interplay between the different types of cells in the brain involves the release and uptake of multiple chemical factors and their resulting signaling cascades in response to stimuli. This creates a constantly changing environment, which complicates the study of the organ’s function. Consequently, dissecting particular aspects of cellular communication is difficult in an in vivo environment. To better dissect particular aspects of cellular communication, controlled in vitro environments are combined with established techniques and novel platforms in this dissertation. Following an introduction to cellular communication and the methods utilized to investigate how it occurs, the development and fabrication of a microfluidic device to examine the interaction of a network of cells is described in chapter 2. This device, composed of a lower pharmacological layer and an upper bulk flow layer, utilizes hydrodynamic focusing to individually address cells without exposing the entire population. Fluorescent and electrochemical data illustrate how the flow streams introduced through the lower layer can be manipulated by fluid sent through the above bulk channels to limit diffusion. In chapter 3, a novel patterning technique, sucrose lift-off lithography, is reported as an alternative to traditional patterning schemes. With this technique, a sacrificial layer of sucrose is initially deposited onto a substrate, which is then coated with photoresist. The resist is then patterned lithographically to expose the substrate in defined well areas. The wells are subsequently loaded with adhesion proteins and a cell suspension, whose culture medium dissolves the sucrose over time, leaving only the patterned cells on the substrate. In chapters 4 and 5, amperometry is utilized to examine the effects of drug treatment and gene modification by knockout on exocytosis. Incubation with the D2 agonist, pramipexole, is found to cause an acute decrease in extracellular calcium influx and a decrease in the amount of dopamine released from in PC12 cells following potassium stimulation in chapter 4. Additionally, the vesicular monoamine transporter (VMAT) is found to package the agonist as well as DA into secretory vesicles. The effects of GODZ and SERZ-β gene knockout on calcium influx and release from intracellular stores in both murine hippocampal neurons and peritoneal mast cells are discussed in Chapter 5. The processes of double knockout neurons, lacking both GODZ and SERZ-β, were found to have decreased calcium influx and release when compared to wild type processes. Moreover, the exocytotic release of histamine and serotonin from mast cells was also examined following stimulation. The release kinetics for both compounds was observed to change with gene deletion as did the number of molecules released from the granules. Loss of GODZ and SERZ-β, whose main function is the posttranslational modification of proteins by palmitoylation, likely causes alterations in the function of proteins that dictate the binding of granules to the plasma membrane. Lastly, pyrolyzed photoresist microelectrodes were characterized for use as a coulometric detector in a microchannel separation device in chapter 6. These pyrolyzed carbon electrodes combined with the short height of the separation channel is able to limit the diffusion of analytes in the separation channel, such that the majority of the analyte that passes over the electrode is oxidized. Following characterization of the system, a mixture of dopamine and serotonin was separated and detected in the device.