Quantitative Measurement of Transmitters in Individual Vesicles in the Cytoplasm of Single Cells with Nanotip Electrodes

Abstract

Quantification of vesicular transmitter contents is important for studying the mechanisms of neurotransmission and malfunction in disease and yet it is incredibly difficult to measure the small contents of neurotransmitters in the attoliter volume of a single vesicle, especially in the cell environment. We introduce a novel method, intracellular vesicle electrochemical cytometry. A nano-tip conical carbon fiber microelectrode is used to electrochemically measure the total contents of electroactive neurotransmitters from individual nanoscale vesicles in single PC12 cells as these vesicles lyse on the electrode inside the living cell. The results demonstrate that only a fraction of quantal contents of neurotransmitter is released during exocytosis. These data support the intriguing hypothesis that the vesicle does not open all the way during the normal exocytosis process, resulting in incomplete expulsion of the vesicular contents.

Nano Secondary Ion Mass Spectrometry Imaging of Dopamine Distribution Across Nanometer Vesicles

“…This value was slightly higher than the previously reported value of 114,300 ± 2105 dopamine molecules 30 and lower than 194,000 ± 13,000 reported by Colliver et al 12 Control PC12 vesicles probed intracellularly contained an average of 152,900 ± 8920 molecules, also higher than previously reported 114,500 ± 15,300 value. 10 In both cases, the difference in measured dopamine content compared with previously reported values might be attributed to differences in culturing and/or experimental conditions. However, we consistently found that the amount released was lower than the amount measured intracellularly in agreement with the concept of partial release.…”

“…The transmitter release upon stimulation was measured with single cell amperometry, 23 whereas dopamine vesicle content was determined with a newly developed technique called intracellular vesicle electrochemical cytometry. 10 Imaging Chemical Content of Single Vesicles by NanoSIMS. We began by chemically imaging the 13 Cdopamine content of LDCVs in PC12 cells.…”

“…Only electrodes with stable and symmetrical cyclic voltammograms were used for the experiments. Cellular electrochemical recordings were performed as previously described, 10 and described above, except the output was filtered at 2 kHz with a low pass 4-pole Bessel filter and digitized at 5 kHz. After data acquisition, each microelectrode was tested in 100 μM dopamine solution, and only data obtained with electrodes with unchanged cyclic voltammogram were used.…”

“…Intracellular cytometry (Figure 1b, middle), introduced by us is a technique to probe the catecholamine content of single vesicles as they lyse on a nanotip microeloectrode inside of a living cell. 10 In both techniques, vesicular dopamine is released and oxidized at the electrode surface resulting in a peak transient. By applying Faraday's law (Q = nNF), it is possible to quantify the oxidation current at the electrode to count the number of dopamine molecules released in moles (N), where Q is the charge that passes through the electrode surface, n is the number of electrons exchanged in a oxidation reaction (2e − for dopamine), and F is Faraday's constant (96,485 Cmol −1 ).…”

“…5−8 Defining the vesicle storage mechanisms would be an important piece of the exocytosis regulation puzzle. 5,9,10 Several cell types have been employed for loading studies among which neuroendocrine cells have had an important role as a model system. They possess two types of secretory vesicles, synaptic-like microvesicles (SLMVs) and so-called large densecore vesicles (LDCVs).…”