Review ArticleRecent developments concerning the investigation of exocytosis with amperometry
Introduction
Chronoamperometry using a single carbon-fiber ultramicroelectrode is nowadays a standard technique for investigating vesicular exocytosis at the single-cell level (see Figure 1). By applying a constant potential at the electrode surface, the released neurotransmitters (e.g. catecholamines) by the emitting cell are oxidized. As a consequence, each vesicular event is displayed as an amperometric spike whose shape features the dynamics and the number of electroactive species released (see Figure 2).
This basic tool from an electroanalytical point of view offers many advantages beyond the monitoring of exocytotic activity. First, ultramicroelectrodes and isolated single cells are globally the same sizes (∼μm). Second, ultramicroelectrodes have a fast time response (∼ms) and a direct current–concentration relationship (i = 4nFDrC∗ where F = 96,485 C mol−1; n = number of electrons, r = electrode radius, C∗ = electroactive species concentration) that allows one to monitor individual vesicular events in terms of real time neurotransmitter fluxes within the electrode-cell cleft. Eventually, the low measured currents (∼pA) and ohmic drop enable to only work with two electrodes (working and reference) which results in easier manipulations at a micrometric level.
Over the past forty years, carbon fiber amperometry (CFA) has significantly contributed to questions related to the exocytotic mechanism [1,2]. First of all, many parameters that govern this important mechanism of neurotransmission were addressed (plasticity, fusion pore, physicochemical and biological assistances) [3]. Second, CFA analytically evolved to be more informative either by means of analytical couplings (fluorescence, electrophysiology) or by being involved at a new scale (arrays, nanoelectrodes) [4, 5, 6, 7]. As a consequence, this short review is aimed at giving the very recent state of the art of this important field. More particularly, only the three past years will be globally considered to ‘take a photograph’ of the field. A broader vision is given by the cited reviews within this article.
Section snippets
CFA as a usual technique to investigate exocytosis
CFA is nowadays used as a standard and compulsory technique to address several issues of exocytosis by biologists. A remaining challenge is to understand how exocytosis is governed by biological processes. First of all, a very interesting result is the unexpected role of endophilins. These proteins usually act as endocytotic adapters, but amperometry and microscopy measurements at chromaffin cells evidence their action as an indispensable vesicular partner to stimulate the priming/fusion of
New analytical tools as an evolution of the CFA technique
Over the past three years, enhancements of the historical CFA configuration were proposed and included many challenges related to the automatic procedure analysis of the amperometric signals, the combination of amperometry and TIRFM (total internal reflection fluorescence microscopy) associated with the search of ideal dual convenient probes, nanoelectrodes, glutamate sensors, microarrays, and electrochemical cytometry (see Figure 3).
Conclusion
This survey of the very recent works devoted to amperometric recordings of vesicular exocytosis is a ‘snapshot’ of a broader field which has started in the 90s. However, this reflects, to some extent, the current state of the art of this research domain. First of all, CFA is still a ‘routine’ technique that is no longer reserved for its only inventors. This positive point means that it is a ‘popular’ technique in vesicular exocytosis similar to NMR in molecular chemistry. This is why CFA still
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work has been supported in part by CNRS (UMR 8640), Ecole Normale Supérieure, French Ministry of Research, Faculté des Sciences et Ingénierie - Sorbonne Université. M. G-C. thanks ‘Emergences Ville de Paris 2014’ Grant and Institut Universitaire de France Junior 2015 Fellowship Program.
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