Donna M. Omiatek, Michael L. Heien, and Andrew G. Ewing


         The fundamental mechanism for neuronal communication, exocytosis, involves the extracellular release of chemical messengers (e.g. neurotransmitters) that are packaged intracellularly in vesicles.  Although a variety of standard analytical methods used to measure stimulus-coupled secretion from single cells have classically been thought to assess the entire content of vesicles, there is evidence in the literature that suggests the total transmitter stored in vesicles is, in fact, not expelled during exocytosis.  To directly interrogate this hypothesis, we have developed a novel technology, termed electrochemical cytometry, to probe the contents of individual biological vesicles extracted from the cell environment. The electrochemical cytometry experimental platform is composed of a hybrid capillary-microfluidic device, which functions to selectively separate, lyse, and quantify the redox-active content of individual vesicles.  This experimental approach allows for the quantification of total vesicular neurotransmitter in a manner that circumvents the biophysical release processes of the cell associated with exocytosis. 

         Direct comparisons of electrochemical data recorded from stimulus-coupled secretion experiments at single cells versus this novel cell-free measurement reveal that, on average, the vesicle releases only a fraction (~40%) of its total transmitter load during exocytosis.  This study provided direct evidence to support the intriguing hypothesis that the mechanism of neurotransmission can be regulated at the single vesicle level.   Moreover, electrochemical cytometry has been applied to the high-throughput investigation of vesicular neurotransmitter loading and depletion as a result of in vivo pharmacological manipulation.  Mice have been injected with various pharmacological agents to augment vesicular neurotransmitter levels, and individual synaptic vesicles extracted from the striata of these mice have been investigated on the electrochemical cytometry platform.  This methodology has served to directly monitor the fundamental mechanisms that outline the efficacies of drugs of abuse (e.g. amphetamine) in the subcellular domain.  Additionally, it has highlighted the vesicle as a potential pharmaceutical target for addressing therapies associated with neurological disease.