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

Abstract

The quantification of vesicular transmitter content is important for studying the mechanisms of neurotransmission and malfunction in disease,a nd yet it is incredibly difficult to measure the tiny amounts of neurotransmitters in the attoliter volume of as ingle vesicle,e specially in the cell environment. We introduce an ovel method, intracellular vesicle electrochemical cytometry.Ananotip conical carbon-fiber microelectrode was used to electrochemically measure the total content of electroactive neurotransmitters in individual nanoscale vesicles in single PC12 cells as these vesicles lysed on the electrode inside the living cell. The results demonstrate that only af raction of the quantal neurotransmitter content is released during exocytosis.T hese data support the intriguing hypothesis that the vesicle does not open all the wayduring the normal exocytosis process,thus resulting in incomplete expulsion of the vesicular contents.

Vesicle impact electrochemical cytometry compared to amperometric exocytosis measurements

“…Inside the cytosol, vesicles undergo the same process as in VIEC; they adsorb, electroporate, and the content is oxidized, which allows quantification of vesicular content inside the natural environment of the cell. Comparison of IVIEC and VIEC measurements performed on the same cell type (PC12) results in similar values, further suggesting that it is the total vesicular content that is detected [20••], although leakage during vesicle preparation might be an issue. It is important to realize that for these measurements IVIEC is a tool for observing the cellular response of a drug or stimulus on the vesicular content, whereas VIEC using isolated vesicles bypasses the cell and the regulatory mechanism of individual vesicles can be studied.…”

“…Li et al combined the IVIEC and SCA techniques to investigate the effect of l -3,4-dihydroxy-phenylalanine ( l -DOPA), which is a biochemical precursor to dopamine, on vesicular content versus exocytosis release from PC12 cells [20••]. 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.…”

“…Average number of catecholamine molecules per vesicle as determined by stimulated exocytosis (blank) versus IVIEC (solid) with (19 cells) or without treatment (17 cells) with l -DOPA (100 mM, 2 h) [20]. Error is the standard error of the mean (SEM).…”

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

“…However, the value of I max and the amount of molecules released are decreased significantly (38%, p = 0.0007 and 28%, p = 0.0018, respectively). The decrease in the amount of molecules released might result from the depletion of vesicular monoamine content or a decrease in the fraction released during exocytosis [30]. The changes observed following BDMC treatment are different from those for curcumin, suggesting curcumin and BDMC influence exocytosis through different mechanisms.…”

Integrated multi-ISE arrays with improved sensitivity, accuracy and precision

“…Furthermore, we demonstrate the successful application of the EIMES for accurate measurement of a tiny change in ion concentration and potentiometric acid base titration. Some researchers have made good experimental progress in electrode miniaturization to date50515253545556. With the development of miniaturized electrodes, the EIMES, a sensitive and universal method for accurate measurement, has potential for widespread use.…”