Vesicular neurotransmitters exocytosis monitored by amperometry: theoretical quantitative links between experimental current spikes shapes and intravesicular structures.

Single cell amperometry has proven to be a powerful and well-established method for characterizing single vesicular exocytotic events elicited at the level of excitable cells under various experimental conditions. Nevertheless, most of the reported characteristics are descriptive, being mostly concerned with the morphological characteristics of the recorded current spikes (maximum current intensities, released charge, rise and fall times, etc.) which are certainly important but do not provide sufficient kinetic information on exocytotic mechanisms due to lack of quantitative models. Here, continuing our previous efforts to provide rigorous models rationalizing the kinetic structures of frequently encountered spike types (spikes with unique exponential decay tails and kiss-and-run events), we describe a new theoretical approach enabling a quantitative kinetic modeling of all types of exocytotic events giving rise to current spikes exhibiting exponential decay tails. This model follows directly from the fact that the condensation of long intravesicular polyelectrolytic strands by high concentrations of monocationic neurotransmitter molecules leads to a matrix structure involving two compartments in constant kinetic exchanges during release. This kinetic model has been validated theoretically (direct and inverse problems) and its experimental interest established by the analysis of the amperometric spikes relative to chromaffin and PC12 cells previously published by some of us.

Electrochemical Storage of Atomic Hydrogen on Single Layer Graphene.

If hydrogen can be stored and carried safely at a high density, hydrogen-fuel cells offer effective solutions for vehicles. The stable chemisorption of atomic hydrogen on single layer graphene (SLG) seems a perfect solution in this regard, with a theoretical maximum storage capacity of 7.7 wt %. However, generating hydrogenated graphene from H2 requires extreme temperatures and pressures. Alternatively, hydrogen adatoms can easily be produced under mild conditions by the electroreduction of protons in solid/liquid systems. Graphene is electrochemically inert for this reaction, but H-chemisorption on SLG can be carried out under mild conditions via a novel Pt-electrocatalyzed "spillover-surface diffusion-chemisorption" mechanism, as we demonstrate using dynamic electrochemistry and isotopic Raman spectroscopy. The apparent surface diffusion coefficient (∼10-5 cm2 s-1), capacity (∼6.6 wt %, ∼85.7% surface coverage), and stability of hydrogen adatoms on SLG at room temperature and atmospheric pressure are significant, and they are perfectly suited for applications involving stored hydrogen atoms on graphene.

Intracellular Electrochemical Nanomeasurements Reveal that Exocytosis of Molecules at Living Neurons is Subquantal and Complex.

Since the early work of Bernard Katz, the process of cellular chemical communication through exocytosis, quantal release, has been considered to be all or none. Recent evidence has shown exocytosis to be partial or "subquantal" at single-cell model systems, but there is a need to understand this at communicating nerve cells. Partial release allows nerve cells to control the signal at the site of release during individual events, for which the smaller the fraction released, the greater the range of regulation. Herein, we show that the fraction of the vesicular octopamine content released from a living Drosophila larval neuromuscular neuron is very small. The percentage of released molecules was found to be only 4.5 % for simple events and 10.7 % for complex (i.e., oscillating or flickering) events. This large content, combined with partial release controlled by fluctuations of the fusion pore, offers presynaptic plasticity that can be widely regulated.

Amperometric Measurements and Dynamic Models Reveal a Mechanism for How Zinc Alters Neurotransmitter Release.

Zinc, a suspected potentiator of learning and memory, is shown to affect exocytotic release and storage in neurotransmitter-containing vesicles. Structural and size analysis of the vesicular dense core and halo using transmission electron microscopy was combined with single-cell amperometry to study the vesicle size changes induced after zinc treatment and to compare these changes to theoretical predictions based on the concept of partial release as opposed to full quantal release. This powerful combined analytical approach establishes the existence of an unsuspected strong link between vesicle structure and exocytotic dynamics, which can be used to explain the mechanism of regulation of synaptic plasticity by Zn2+ through modulation of neurotransmitter release.

Electrochemical Monitoring of ROS/RNS Homeostasis Within Individual Phagolysosomes Inside Single Macrophages.

The existence of a homeostatic mechanism regulating reactive oxygen/nitrogen species (ROS/RNS) amounts inside phagolysosomes has been invoked to account for the efficiency of this process but could not be unambiguously documented. Now, intracellular electrochemical analysis with platinized nanowire electrodes (Pt-NWEs) allowed monitoring ROS/RNS effluxes with sub-millisecond resolution from individual phagolysosomes impacting onto the electrode inserted inside a living macrophage. This shows for the first time that the consumption of ROS/RNS by their oxidation at the nanoelectrode surface stimulates the production of significant ROS/RNS amounts inside phagolysosomes. These results establish the existence of the long-postulated ROS/RNS homeostasis and allows its kinetics and efficiency to be quantified. ROS/RNS concentrations may then be maintained at sufficiently high levels for sustaining proper pathogen digestion rates without endangering the macrophage internal structures.

Self-Inhibitory Electron Transfer of the Co(III)/Co(II)-Complex Redox Couple at Pristine Carbon Electrode.

Applications of conducting carbon materials for highly efficient electrochemical energy devices require a greater fundamental understanding of heterogeneous electron-transfer (ET) mechanisms. This task, however, is highly challenging experimentally, because an adsorbing carbon surface may easily conceal its intrinsic reactivity through adventitious contamination. Herein, we employ nanoscale scanning electrochemical microscopy (SECM) and cyclic voltammetry to gain new insights into the interplay between heterogeneous ET and adsorption of a Co(III)/Co(II)-complex redox couple at the contamination-free surface of electron-beam-deposited carbon (eC). Specifically, we investigate the redox couple of tris(1,10-phenanthroline)cobalt(II), Co(phen)32+, as a promising mediator for dye-sensitized solar cells and redox flow batteries. A pristine eC surface overlaid with KCl is prepared in vacuum, protected from contamination in air, and exposed to an ultrapure aqueous solution of Co(phen)32+ by the dissolution of the protective KCl layer. We employ SECM-based nanogap voltammetry to quantitatively demonstrate that Co(phen)32+ is adsorbed on the pristine eC surface to electrostatically self-inhibit outer-sphere ET of nonadsorbed Co(phen)33+ and Co(phen)32+. Strong electrostatic repulsion among Co(phen)32+ adsorbates is also demonstrated by SECM-based nanogap voltammetry and cyclic voltammetry. Quantitatively, self-inhibitory ET is characterized by a linear decrease in the standard rate constant of Co(phen)32+ oxidation with a higher surface concentration of Co(phen)32+ at the formal potential. This unique relationship is consistent not with the Frumkin model of double layer effects, but with the Amatore model of partially blocked electrodes as extended for self-inhibitory ET. Significantly, the complicated coupling of electron transfer and surface adsorption is resolved by combining nanoscale and macroscale voltammetric methods.

'Full fusion' is not ineluctable during vesicular exocytosis of neurotransmitters by endocrine cells.

Vesicular exocytosis is an essential and ubiquitous process in neurons and endocrine cells by which neurotransmitters are released in synaptic clefts or extracellular fluids. It involves the fusion of a vesicle loaded with chemical messengers with the cell membrane through a nanometric fusion pore. In endocrine cells, unless it closes after some flickering ('Kiss-and-Run' events), this initial pore is supposed to expand exponentially, leading to a full integration of the vesicle membrane into the cell membrane-a stage called 'full fusion'. We report here a compact analytical formulation that allows precise measurements of the fusion pore expansion extent and rate to be extracted from individual amperometric spike time courses. These data definitively establish that, during release of catecholamines, fusion pores enlarge at most to approximately one-fifth of the radius of their parent vesicle, hence ruling out the ineluctability of 'full fusion'.

On the mechanism of electrochemical vesicle cytometry: chromaffin cell vesicles and liposomes.

The mechanism of mammalian vesicle rupture onto the surface of a polarized carbon fiber microelectrode during electrochemical vesicle cytometry is investigated. It appears that following adsorption to the surface of the polarized electrode, electroporation leads to the formation of a pore at the interface between a vesicle and the electrode and this is shown to be potential dependent. The chemical cargo is then released through this pore to be oxidized at the electrode surface. This makes it possible to quantify the contents as it restricts diffusion away from the electrode and coulometric oxidation takes place. Using a bottom up approach, lipid-only transmitter-loaded liposomes were used to mimic native vesicles and the rupture events occurred much faster in comparison with native vesicles. Liposomes with added peptide in the membrane result in rupture events with a lower duration than that of liposomes and faster in comparison to native vesicles. Diffusional models have been developed and suggest that the trend in pore size is dependent on soft nanoparticle size and diffusion of the content in the nanometer vesicle. In addition, it appears that proteins form a barrier for the membrane to reach the electrode and need to move out of the way to allow close contact and electroporation. The protein dense core in vesicles matrixes is also important in the dynamics of the events in that it significantly slows diffusion through the vesicle.

The evidence for open and closed exocytosis as the primary release mechanism.

Exocytosis is the fundamental process by which cells communicate with each other. The events that lead up to the fusion of a vesicle loaded with chemical messenger with the cell membrane were the subject of a Nobel Prize in 2013. However, the processes occurring after the initial formation of a fusion pore are very much still in debate. The release of chemical messenger has traditionally been thought to occur through full distention of the vesicle membrane, hence assuming exocytosis to be all or none. In contrast to the all or none hypothesis, here we discuss the evidence that during exocytosis the vesicle-membrane pore opens to release only a portion of the transmitter content during exocytosis and then close again. This open and closed exocytosis is distinct from kiss-and-run exocytosis, in that it appears to be the main content released during regular exocytosis. The evidence for this partial release via open and closed exocytosis is presented considering primarily the quantitative evidence obtained with amperometry.

Enhancing the Bipolar Redox Cycling Efficiency of Plane-Recessed Microelectrode Arrays by Adding a Chemically Irreversible Interferent.

The individual electrochemical anodic responses of dopamine (DA), epinephrine (EP), and pyrocatechol (CT) were investigated at arrays of recessed gold disk-microelectrodes arrays (MEAs) covered by a gold plane electrode and compared to those of their binary mixture (CT and EP) when the top-plane electrode was operated as a bipolar electrode or as a collector. The interferent species (EP) displays a chemically irreversible wave over the same potential range as the chemically reversible ones of DA or CT. As expected, in the generator-collector (GC) mode, EP did not contribute to the redox cycling amplification that occurred only for DA or CT. Conversely, in the bipolar mode, the presence of EP drastically increased the bipolar redox cycling efficiency of DA and CT. This evidenced that the chemically irreversible oxidation of EP at the anodic poles of the top plane floating electrode provided additional electron fluxes that were used to more efficiently reduce the oxidized DA or CT species at the cathodic poles. This suggests an easy experimental strategy for enhancing the bipolar efficiency of MEAs up to reach a performance identical to that achieved when the same MEAs are operated in a GC mode.

Electrochemical Measurements of Optogenetically Stimulated Quantal Amine Release from Single Nerve Cell Varicosities in Drosophila Larvae.

The nerve terminals found in the body wall of Drosophila melanogaster larvae are readily accessible to experimental manipulation. We used the light-activated ion channel, channelrhodopsin-2, which is expressed by genetic manipulation in Type II varicosities to study octopamine release in Drosophila. We report the development of a method to measure neurotransmitter release from exocytosis events at individual varicosities in the Drosophila larval system by amperometry. A microelectrode was placed in a region of the muscle containing a varicosity and held at a potential sufficient to oxidize octopamine and the terminal stimulated by blue light. Optical stimulation of Type II boutons evokes exocytosis of octopamine, which is detected through oxidization at the electrode surface. We observe 22700±4200 molecules of octopamine released per vesicle. This system provides a genetically accessible platform to study the regulation of amine release at an intact synapse.

Real-time Monitoring of Discrete Synaptic Release Events and Excitatory Potentials within Self-reconstructed Neuromuscular Junctions.

Chemical synaptic transmission is central to the brain functions. In this regard, real-time monitoring of chemical synaptic transmission during neuronal communication remains a great challenge. In this work, in vivo-like oriented neural networks between superior cervical ganglion (SCG) neurons and their effector smooth muscle cells (SMC) were assembled in a microfluidic device. This allowed amperometric detection of individual neurotransmitter release events inside functional SCG-SMC synapse with carbon fiber nanoelectrodes as well as recording of postsynaptic potential using glass nanopipette electrodes. The high vesicular release activities essentially involved complex events arising from flickering fusion pores as quantitatively established based on simulations. This work allowed for the first time monitoring in situ chemical synaptic transmission under conditions close to those found in vivo, which may yield important and new insights into the nature of neuronal communications.

Strategy for increasing the electrode density of microelectrode arrays by utilizing bipolar behavior of a metallic film.

Recessed microelectrode arrays and plane-recessed microelectrode arrays (MEAs) with different center-to-center distances are designed and fabricated using lithographic technology. By comparing electrochemical behavior of plane-recessed MEAs with that of recessed MEAs, bipolar phenomenon of the metallic plane film is revealed. Redox cycling can occur when the top plane electrode was floating; that is, the bipolar behavior of the unbiased top plane electrode may perform locally as a collector and enlarge the concentration gradient of Ru(NH3)6Cl3 and thus promote an apparent generator/collector electrochemical response of the microdisk electrode in the MEAs configuration. By utilizing the bipolar behavior, the center-to-center distance of MEAs required for achieving steady-state current could decrease without favoring at the same time the overlapping of diffusion layers of microelectrodes, and thus, the electrode density of MEAs can be increased. Therefore, the bipolar behavior of the metallic film can increase both the current response of an individual microdisk and the electrode density of microdisks without losing the characteristics of a microelectrode. By just fabricating a thin layer of metallic film on the plane and leaving it floating without potential control, recessed MEAs used in this work can achieve the increase of detection sensitivity by more than 1 order of magnitude.

Theoretical investigation of generator-collector microwell arrays for improving electroanalytical selectivity: application to selective dopamine detection in the presence of ascorbic acid.

Recessed generator-collector assemblies consisting of an array of recessed disks (generator electrodes) with a gold layer (collector electrode) deposited over the top-plane insulator reportedly allow increased selectivity and sensitivity during electrochemical detection of dopamine (DA) in the presence of ascorbic acid (AA), a situation which is frequently encountered. In sensor design, the potential of the disk electrodes is set to the wave plateau of DA, whereas the plane electrode is biased at the irreversible wave plateau of AA before the onset of the DA oxidation wave. Thus, AA is scavenged but DA is allowed to enter the nanocavities to be oxidized at the disk electrodes, and its signal is further amplified by redox cycling between disk and plane electrodes. Several different theoretical approaches are elaborated herein to analyze the behavior of the system, and their conclusions are successfully tested by experiments. This reveals the crucial role of the plane-electrode area which screens access to the recessed disks (i.e. acts as a diffusional Faraday cage) and simultaneously contributes to amplification of the analyte signal through positive feedback, as occurs in interdigitated arrays and scanning electrochemical microscopy. Simulations also allow for the evaluation of the benefits of different geometries inspired by the above design and different operating modes for increasing the sensor performance.

Vesicular release of neurotransmitters: converting amperometric measurements into size, dynamics and energetics of initial fusion pores.

Amperometric currents displaying a pre-spike feature (PSF) may be treated so as to lead to precise information about initial fusion pores, viz., about the crucial event initiating neurotransmitter vesicular release in neurons and medullary glands. However, amperometric data alone are not self-sufficient, so their full exploitation requires external calibration to solve the inverse problem. For this purpose we resorted to patch-clamp measurements published in the literature on chromaffin cells. Reported pore radii were thus used to evaluate the diffusion rate of neurotransmitter cations in the partially altered matrix located near the fusion pore entrance. This allowed an independent determination of each initial fusion pore radius giving rise to a single PSF event. The statistical distribution of the radii thus obtained provided for the first time an experimental access to the potential energy well governing the thermodynamics of such systems. The shape of the corresponding potential energy well strongly suggested that, after their creation, initial fusion pores are essentially controlled by the usual physicochemical laws describing pores formed in bilayer lipidic biological membranes, i.e., they have an essentially lipidic nature.

A novel approach to the simulation of electrochemical mechanisms involving acute reaction fronts at disk and band microelectrodes.

A new simulation algorithm is presented for describing the dynamics of diffusion reactions at the most common microelectrode 1D (planar, cylindrical, spherical) and 2D geometries (band, disk) for electrochemical mechanisms of any complexity and involving fast homogeneous reactions of any kind. A series of typical electrochemical mechanisms that create the most severe simulation difficulties is used to establish the exceptional performance and accuracy of this algorithm, which stem from the combination of (quasi)conformal transformation of space and a new method for auto-adaptive grid compression.

Simple and clear evidence for positive feedback limitation by bipolar behavior during scanning electrochemical microscopy of unbiased conductors.

On the basis of an experimentally validated simple theoretical model, it is demonstrated unambiguously that when an unbiased conductor is probed by a scanning electrochemical tip (scanning electrochemical microscopy, SECM), it performs as a bipolar electrode. Though already envisioned in most recent SECM theories, this phenomenon is generally overlooked in SECM experimental investigations. However, as is shown here, this may alter significantly positive feedback measurements when the probed conductor is not much larger than the tip.

Diffusion from within a spherical body with partially blocked surface: diffusion through a constant surface area.

Diffusion from spherical bodies has been a subject of interest since the earliest times of modern sciences and a few equivalent analytical formulations of the problem are taught in engineering textbooks dealing with cooling rates of hot spheres. However, all these former studies assume that the diffusing material is transferable to/from the surrounding space through the whole surface of the spherical body. Conversely, the development of nanoscience and the improved knowledge of microscopic biological events have evidenced that diffusion from spherical bodies is a ubiquitous problem. It often occurs in situations where the nanosphere surfaces are not isotropic and partly impermeable to diffusing materials. This work elaborates on this issue and theoretically establishes that--with some specific allowance--the basic analytical equation of diffusion from/to fully accessible spherical bodies may be used.

Reconstruction of aperture functions during full fusion in vesicular exocytosis of neurotransmitters.

Individual vesicular exocytosis of adrenaline by dense core vesicles in chromaffin cells is considered here as a paradigm of many situations encountered in biology, nanosciences and drug delivery in which a spherical container releases in the external environment through gradual uncovering of its surface. A procedure for extracting the aperture (opening) function of a biological vesicle fusing with a cell membrane from the released molecular flux of neurotransmitter as monitored by amperometry has been devised based on semi-analytical expressions derived in a former work [C. Amatore, A. I. Oleinick, I. Svir, ChemPhysChem 2009, 10, DOI: 10.1002/cphc.200900646]. This precise analysis shows that in the absence of direct information about the radius of the vesicle or about the concentration of the adrenaline cation stored by the vesicle matrix, current spikes do not contain enough information to determine the maximum aperture angle. Yet, a statistical analysis establishes that this maximum aperture angle is most probably less than a few tens of degrees, which suggests that full fusion is a very improbable event.

Capacitive and solution resistance effects on voltammetric responses at a disk microelectrode covered with a self-assembled monolayer in the presence of electron hopping.

This article extends our previous works (Amatore, C.; Oleinick, A.; Svir, I. Anal. Chem. 2008, 80, 7947-7956; 7957-7963.) about the effects of resistive and capacitive distortions in voltammetry at disk microelectrodes. The particular case of voltammetry of a self-assembled monolayer carrying one redox site per molecule is investigated here. In addition, the effect of an uneven distribution of the effective electrochemical potential on the possibility of electron hopping (EH) contributions is examined. An original model of EH has been developed considering both diffusion-type (i.e., related to concentration gradients) and migration-type (i.e., imposed by an uneven distribution of the electrical potential due to an ohmic drop and capacitance charging) contributions. This predicts that as soon as the system performs out of thermodynamic equilibrium and provided that the EH rate constants are not too small the system tends to re-establish its out-of-equilibrium state through EH. Hence, EH somewhat tries to compensate the voltammetric distortions that would be enforced by the uneven distribution of the electrochemical driving force incurred by the system due to an ohmic drop and capacitive charging. However, this rigorous analysis established that, though EH may be effective under specific circumstances particularly near the electrode edge, its overall influence on voltammetric waves remains negligible for any realistic experimental situation.