ABSTRACT
Membrane fusion involves the merging of two phospholipid bilayers in an aqueous environment and is involved in many cellular processes, such as cell exocytosis. Synaptic vesicles are nanometersized organelles, which are packaged with chemical messengers (e.g., neurotransmitters, neurohormones, and neuropeptides).1-3 Each presynaptic nerve terminal contains hundreds of synaptic vesicles. When an action potential depolarizes the presynaptic plasma membrane, Ca2+ channels
14.1 Introduction .......................................................................................................................... 513 14.2 Spatial and Temporal Resolution of Cell Exocytosis Studied by Individually
Addressable Subcelluar Size MEAs ..................................................................................... 515 14.2.1 Spatially and Temporally Resolved Single-Cell Exocytosis Measured with
Individually Addressable Carbon-Fiber MEAs ........................................................ 515 14.2.2 Spatially and Temporally Resolved Single-Cell Exocytosis
Utilizing Individually Addressable Carbon-Ring MEAs ......................................... 517 14.2.3 Spatially and Temporally Resolved Single-Cell Exocytosis
Utilizing Individually Addressable Thin-Film MEAs ............................................. 520 14.2.4 Concurrent Events Detected at Single Cells with Different
Electrodes of the MEA ............................................................................................. 524 14.3 Cell Mimic Systems for Exocytosis...................................................................................... 526
14.3.1 Study of Exocytosis with an Articial Cell Model Using Liposomes and Lipid Nanotubes ................................................................................................. 526
14.3.2 Study of Exocytosis with an Articial Cell Model Using a DNA Zipper as a SNARE-Protein Mimic ............................................................................................ 528
14.3.3 Study of Exocytosis with an Articial Cell Model Constructed from PC12 Cell Plasma Membrane Vesicles (Blebs) ......................................................... 530
14.4 Future Perspectives ............................................................................................................... 533 Acknowledgments .......................................................................................................................... 533 References ...................................................................................................................................... 534
open, and Ca2+ ows into the nerve terminal to trigger the exocytosis of synaptic vesicles, thereby vesicles migrate to the plasma membrane of a cell, fuse, and release their contents into the extracellular space. These messengers can then bind to receptors on a target cell, thus inducing a cascade of signaling events in a complex network3,4 (Figure 14.1). Until now, several types of neurotransmitters, such as amino acids, monoamines, and peptides, have been found, but their function in the brain is not always clear. Exocytotic events occur on a millisecond timescale with transmitter release proportions varying from zepto-to femtomole amounts per vesicle,5 making them experimentally challenging to monitor. Several different kinds of bioanalytical techniques have been developed to measure chemical messengers in the extracellular uid following exocytosis from tissue in vivo and to measure individual exocytotic events at single cells under in vitro experimental conditions with biological or articial models.6-21
Electrochemical methods utilizing carbon-ber microelectrodes have been extremely useful in the detection of neurotransmitters released from single vesicles.7,8 In a typical experiment, a carbon-ber microdisk electrode is placed on the cell surface. The electrode potential is held constant (in amperometry) or scanned (in cyclic voltammetry) with respect to a reference electrode placed in the extracellular media. Quantitative and qualitative information about neurotransmission can be obtained from i-t and i-V responses.22 Experiments using a single carbon-ber microelectrode provide information including insights into chemical identity, amount of neurotransmitter released from a single vesicle, event frequency, and kinetic information relating to fusion pore opening.23 However, the specialized protein machineries and lipid domains in the cell membrane lead to spatial variations in the cell membranes as well as the nature and location of exocytotic release.24,25 For example, the distribution of exocytotic activity has been found to be spatially heterogeneous at the surface of a single cell.26-30 Such information can be useful in understanding the molecular mechanisms and the chemical basis for the regulation of neural secretion. Due to the difculties in micromanipulation, it is extremely challenging to record from more than one microelectrode concurrently at a single cell.
