Quantitative Chemical Measurements of Vesicular Transmitters with Electrochemical Cytometry

Abstract

Electrochemical cytometry adds a new dimension to our ability to study the chemistry and chemical storage of transmitter molecules stored in nanometer vesicles. The approach involves the adsorption and subsequent rupture of vesicles on an electrode surface during which the electroactive contents are quantitatively oxidized (or reduced). The measured current allows us to count the number of molecules in the vesicles using Faraday's law and to correlate this to the amount of molecules released when single exocytosis events take place at communicating cells. The original format for this method involved a capillary electrophoresis separation step to singly address each vesicle, but we have more recently discovered that cellular vesicles tend to adsorb to carbon electrodes and spontaneously as well as stochastically rupture to give mostly single vesicle events. This approach, called impact electrochemical cytometry, even though the impact is perhaps not the important part of this process, has been studied and the vesicle rupture appears to be at the interface between the vesicle and the electrode and is probably driven by electroporation. The pore size and rate of content electrolysis are a function of the pore diameter and the presence of a protein core in the vesicles. In model liposomes with no protein, events appear extremely rapidly as the soft nanoparticles impact the electrode and the contents are oxidized. It appears that the proteins decorating the surface of the vesicle are important in maintaining a gap from the electrode and when this gap is closed electroporation takes place. Models of the event response times suggest the pores formed are small enough so we can carry out these measurements at nanotip electrodes and we have used this to quantify the vesicle content in living cells in a mode we call intracellular impact electrochemical cytometry. The development of electrochemical cytometry allows comparison between vesicle content and vesicular release and we have found that only part of the vesicle content is released in typical exocytotic cases measured by amperometry. This has led to the novel hypothesis that most exocytosis from dense core vesicles is via mechanism where vesicles fuse with the cell membrane, some content is released and then close again to be reloaded and reused. It leaves open the possibility that cells regulate release during individual events. This might be important in learning and memory and be a nonreceptor pharmaceutical target for brain-related disorders. Indeed, the concept of the chemo-brain observed in cisplatin-treated cancer patients appears to be at least in part the result of changing the fraction of transmitter released and we have been able to show this by using the combined amperometric measurement of release and electrochemical cytometry at model cells.

Vesicle impact electrochemical cytometry compared to amperometric exocytosis measurements

“…This was carried out with the same electrode for the two techniques so as to maintain the same signal to noise ratios for similar measurements and comparison (Figure 3) shows that the vesicle content is considerably larger than the amount released. This supports the partial release hypothesis for amperometrically measured exocytosis where some events open fully, but during most normal events the vesicle fuses, opens, and then closes again as is illustrated in for the different modes in Figure 4 [21•,22••]. Cells were also incubated with HEPES physiological saline containing 100 µM l -DOPA for 2 h and then rinsed three times with HEPES physiological saline prior to the experiments.…”

Analytical Techniques in Neuroscience: Recent Advances in Imaging, Separation, and Electrochemical Methods

“…148–151 To quantify the amount of neurotransmitter in a vesicle, the Ewing group developed a new microfluidic device with electrochemical detection, called electrochemical cytometry. 152 This is a hybrid microfluidic-capillary electrophoresis-electrochemical detection platform that separates individual vesicles by capillary electrophoresis so that vesicles elute one at a time from the channel. An electrode at the end detects neurotransmitters from each vesicle as they break in contact with it.…”

Single cell amperometry reveals curcuminoids modulate the release of neurotransmitters during exocytosis from PC12 cells

“…In the open and closed mechanism of exocytosis, only a fraction of neurotransmitters is released during normal exocytosis [30,36,37,38]. For some transmitters to remain in the vesicle after exocytosis, the rate of fusion pore constriction has to be faster than the diffusion rate of neurotransmitters coming out of the vesicle [20,21].…”