Figure 1 - uploaded by David Sulzer
Content may be subject to copyright.
An overview of amperometric data acquisition and analysis.The original 'true' amperometric signal is ultimately represented by spike characteristics pooled and analyzed from several recordings. During the processing of individual traces, each analytical step may introduce some signal distortion, depending on the internal characteristics of the recording (signal frequencies, noise level and others) and on user-defined parameters of the analysis (digital filters, detection thresholds, cutoffs and others).
Source publication
Amperometry is widely used to study exocytosis of neurotransmitters and hormones in various cell types. Analysis of the shape of the amperometric spikes that originate from the oxidation of monoamine molecules released during the fusion of individual secretory vesicles provides information about molecular steps involved in stimulation-dependent tra...
Contexts in source publication
Context 1
... problems arise when dI/dt is used for event detection. First, in a typical amperometric trace, differentiation decreases the signal-to-noise ratio thereby demanding additional fil- tering of the dI/dt (Box 1). Second, this method may perform poorly when determining the baseline of spikes that have PSF with very long steady-state signals (see next). ...
Context 2
... the presence of high- frequency noise complicates the search for the timepoint when the transmitter concentration declines to the initial levels. Although some procedures can improve proper baseline positioning ( Supplementary Fig. 1 online and Supplementary Methods), visual trace examination and baseline readjustment are often necessary. ...
Context 3
... together, these changes are characteristic of release events that originate far away from the electrode and undergo 'dif- fusional filtering' , similar to the subpopulation of spikes that have longer t rise values 13 . Supporting this idea, the incidence of observing a foot was higher for spikes that had shorter t rise or double-expo- nential falling phases (Supplementary Table 1). Nevertheless, spikes with and without the PSF had similar durations and risetimes, argu- ing against a possibility that the foot can be observed exclusively within the subpopulation of release events occurring closest to the electrode (Supplementary Fig. 2 online). ...
Context 4
... discussed, spike parameters can be distorted by 'diffusional fil- tering' , and thus maximal t 1/2 or t rise are sometimes used as cutoffs to eliminate 'slow' spikes 13,22 (Supplementary Table 1). Our data also suggest that the shape of the spike falling phase might be indicative of the proximity of the release site to the electrode surface, although alternative explanations, such as the presence of different vesicle pop- ulations cannot be excluded. ...
Context 5
... all feet are analyzed, the amplitude of PSFs that do not reach steady state cannot be measured directly and therefore is resolved as an average current within t foot (b, horizontal solid line). If only PSFs with steady states are analyzed (see Supplementary Fig. 1 for the methodology of finding the steady state), I foot can be determined with better precision using an averaged current on the PSF plateau (c) or an exponential fit of the trace from T bkg1 to the end of the foot 22 (a, dotted curved line). ...
Citations
... Each of these spikes represented the oxidation of catecholamine molecules released by single vesicles through exocytosis, and they unambiguously demonstrated that mechanical strain could induce catecholamine release from chromaffin cell upon loading. To explore the kinetic information of strain-evoked exocytotic events, series of featured parameters of spikes were analyzed, including peak current (I max ) relevant to the geometrical size of fusion pore, number of released molecules (N) given by their electrical charges (Q), t 1/2 indicating the duration of exocytosis, t rise and t fall representing the opening and closing time of fusion pore [12,23] (Figure 4B Figure S14 and Table S1). ...
Exocytosis involving the fusion of intracellular vesicles with cell membrane, is thought to be modulated by the mechanical cues in the microenvironment. Single‐cell electrochemistry can offer unique information about the quantification and kinetics of exocytotic events; however, the effects of mechanical force on vesicular release have been poorly explored. Herein, we developed a stretchable microelectrode with excellent electrochemical stability under mechanical deformation by microfabrication of functionalized poly(3,4‐ethylenedioxythiophene) conductive ink, which achieved real‐time quantitation of strain‐induced vesicular exocytosis from a single cell for the first time. We found that mechanical strain could cause calcium influx via the activation of Piezo1 channels in chromaffin cell, initiating the vesicular exocytosis process. Interestingly, mechanical strain increases the amount of catecholamines released by accelerating the opening and prolonging the closing of fusion pore during exocytosis. This work is expected to provide revealing insights into the regulatory effects of mechanical stimuli on vesicular exocytosis.
... 14 Amperometry also allows the analysis of the dynamics of catecholamines release from individual exocytosis events (Mosharov and Sulzer, 2005). To determine whether A618T or S619L affect the characteristics of the individual exocytotic events, we further analyzed different spike parameters (cartoon on Fig. 4 A), particularly the spike charge (Q), that is proportional to the amount of catecholamines released during each individual event, the spike amplitude, which accounts for the height of the spike, and time elapsed at spike´s half amplitude (HAT). ...
Dynamins are large GTPases whose primary function is to catalyze membrane scission during endocytosis, but also modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin-2 mutations, p.A618T and p.S619L, impair Ca ²⁺ -induced exocytosis of GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high-temporal resolution techniques, such as patch-clamp capacitance measurements and carbon-fiber amperometry to assess the effects of these mutations on these two cellular processes using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin-2 mutants inhibits a dynamin and F-actin dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin-2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of NPY-labelled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with DMPP. They also provoked a significant decrease in the Ca ²⁺ -induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the CNM- linked p.A618T and p.S619L mutations in dynamin-2 affect exocytosis and endocytosis, being the disruption of F-actin a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations.
... This technique has a high sensitivity, allowing the estimation o released molecules by individual vesicles since the intensity of the signal is proportiona to the collected electrons. The high temporal resolution allows the quantification of the spike kinetic parameters [10]. The main steps of vesicular exocytosis after packing monoamines into vesicles begin with docking to the plasma membrane, vesicle priming and the formation of fusion pores. ...
... Trials include monitoring the spontaneous exocytotic activity under control conditions and after L-3,4-dihydroxyphenylalanine (L-DOPA) [26] administration. Acquired data were analyzed using the APE code and compared to Quanta Analysis (Igor macro) by Mosharov et al. [10] to validate the developed software. ...
... This code has been designed to be user-friendly, with a clean interface for data entry and storage of output reports in ASCII, allowing high compatibility with other analysis software. The methods for extracting spike parameters are compatible with strategies proposed in the literature and have already been implemented in other software [10]. ...
MicroGraphited-Diamond-Multi Electrode Arrays (μG-D-MEAs) can be successfully used to reveal, in real time, quantal exocytotic events occurring from many individual neurosecretory cells and/or from many neurons within a network. As μG-D-MEAs arrays are patterned with up to 16 sensing microelectrodes, each of them recording large amounts of data revealing the exocytotic activity, the aim of this work was to support an adequate analysis code to speed up the signal detection. The cutting-edge technology of microGraphited-Diamond-Multi Electrode Arrays (μG-D-MEAs) has been implemented with an automated analysis code (APE, Amperometric Peak Analysis) developed using Matlab R2022a software to provide easy and accurate detection of amperometric spike parameters, including the analysis of the pre-spike foot that sometimes precedes the complete fusion pore dilatation. Data have been acquired from cultured PC12 cells, either collecting events during spontaneous exocytosis or after L-DOPA incubation. Validation of the APE code was performed by comparing the acquired spike parameters with those obtained using Quanta Analysis (Igor macro) by Mosharov et al.
... Several well-structured programs that facilitate the analysis of such data exist in the electrochemistry community, the most commonly used being IgorPro QuantaAnalysis software. 5 QuantaAnalysis is a mature software dedicated to the 4 analysis of amperometry traces that allows digital filtering and analysis of the current noise, spike identification, calculation of over 20 spike kinetic parameters, and visualization. The most commonly measured spike characteristics include the area under the spike (such as Q spike or Q foot ), spike maximal height (I max ) and spike width (t1 2 ) at half its height (where ...
Amperometry is a commonly used electrochemical method for studying the process of exocytosis in real-time. Given the high precision of recording that amperometry procedures offer, the volume of data generated can span over several hundreds of megabytes to a few gigabytes and therefore necessitates systematic and reproducible methods for analysis. Though the spike characteristics of amperometry traces in the time domain hold information about the dynamics of exocytosis, these biochemical signals are, more often than not, characterized by time-varying signal properties. Such signals with time-variant properties may occur at different frequencies and therefore analyzing them in the frequency domain may provide statistical validation for observations already established in the time domain. This necessitates the use of time-variant, frequency-selective signal processing methods as well, which can adeptly quantify the dominant or mean frequencies in the signal. The Fast Fourier Transform (FFT) is a well-established computational tool that is commonly used to find the frequency components of a signal buried in noise. In this work, we outline a method for spike-based frequency analysis of amperometry traces using FFT that also provides statistical validation of observations on spike characteristics in the time domain. We demonstrate the method by utilizing simulated signals and by subsequently testing it on diverse amperometry datasets generated from different experiments with various chemical stimulations. To our knowledge, this is the first fully automated open-source tool available dedicated to the analysis of spikes extracted from amperometry signals in the frequency domain.
... The oxidation product of CA released from a single secretory granule will be detected as amperometric spikes. The amperometric individual spike can be identified, measured, and analyzed to obtain a statistical analysis of frequency, amplitude, and kinetics parameters [32,33]. ...
... 5. Verify each spike selected by the Quanta Analysis application [32]; it allows to add or delete spikes with a high magnification view. ...
... 17. For more detailed information about the amperometric spike parameters, these articles can be consulted: Mosharov and Sulzer [32], Segura et al. [33]. ...
The spontaneously hypertensive rat (SHR) is a model widely used to investigate the causal mechanisms of essential hypertension. The enhanced catecholamine (CA) release reported in adrenal glands from adult SHRs raised considerable interest for its possible implication in the genesis of hypertension. The use of powerful techniques such as calcium imaging, electrophysiology, and single-cell amperometry to monitor in real time the key steps in CA secretion has allowed a better understanding of the role of chromaffin cells (CC) in the pathophysiology of hypertension, although several questions remain. Additionally, the implementation of these techniques in preparations in situ, such as the acute adrenal gland slice, which maintains the microenvironment, cell-to-cell communication, and anatomical structure similar to that of the intact adrenal gland, yields data that may have even greater physiological relevance. Here, we describe the procedures to measure the blood pressure of rats in a noninvasive manner, how to obtain primary cultures of adrenal chromaffin cells and acute adrenal slices, and how to perform amperometric recordings and intracellular calcium imaging in these preparations.Key wordsChromaffin cellsAdrenal slicesCalcium imagingElectrophysiologyAmperometryHypertension
... The purpose of this study is to investigate the carrier generation when the enzyme chitinase is applied to the chitin membrane. The generation and diffusion of carriers can be studied by various methods, but in this study, we will use amperometry, which is simple and quick to measure [32][33][34][35]. In this study, we use amperometry, which is a simple and quick method. ...
Hydrogen energy is focused on as next-generation energy without environmental load. Therefore, hydrogen production without using fossil fuels is a key factor in the progress of hydrogen energy. In the present work, it was found that chitin–chitinase and collagen–collagenase composites can generate protons by the hydrolysis of the enzyme. The concentration of the generated proton in the chitin–chitinase and collagen–collagenase composites are 1.68 × 1017 cm−3 and 1.02 × 1017 cm−3, respectively. Accompanying these results, proton diffusion constants in the chitin and collagen membranes are also estimated to be 8.59 × 10−8 cm2/s and 8.69 × 10−8 cm2/s, respectively. Furthermore, we have fabricated the bio-fuel cell using these composites as hydrogen fuel and demonstrated that these composites become a fuel of the fuel cell.
... Values for I foot , t foot , N molecules (foot), and N foot /N events decrease significantly after the exposure of chaotropic anions (e.g., ClO 4 − ) as shown in Figure S5 (p values are listed in Tables S8−S11). The value for I foot decreases (ClO 4 − ) or remains constant (SCN − ) after exposure of chaotropic anions, even though the vesicle content increases simultaneously, suggesting a smaller initial pore, 46 which could be induced by a tighter actin network than after exposure to Cl − . Moreover, the decreased t foot , N foot /N events , and N molecules in the foot indicate that the chaotropic anions induce a less stable fusion pore, which is consistent with the results from the main peak. ...
Hofmeister effects have often been ignored in living organisms, although they affect the activity and functions of biological molecules. Herein, amperometry has been applied to show that the vesicular content, dynamics of exocytosis and vesicles opening, depend on the anionic species treatment. Compared to 100 μM Cl- treated chromaffin cells, a similar number of catecholamine molecules is released after chaotropic anions (ClO4- and SCN-) treatment, even though the vesicular catecholamine content significantly increases, suggesting a lower release fraction. In addition, there are opposite effects on the dynamics of vesicles release (shorter duration) and vesicle opening (longer duration) for chaotropic anions treated cells. Our results show anion-dependent vesicle release, vesicle opening, and vesicular content, providing understanding of the pharmacological and pathological processes induced by inorganic ions.
... For this reason, the voltammograms of the analyte should be taken by normal (DC) polarography or CV the appropriate plateau region should be selected. [230] Amperometric methods are mostly preferred as sensors and detectors in chromatographic devices. Also chronoamperometry is a time-dependent technique where a square-wave potential is applied to the working electrode. ...
... Furthermore, these are a very sensitive methods since a unique tension is applied to the analyte. [228][229][230] Chronoamperometry is a technique which does not require labeling of the analyte or bioreceptor and has been applied in many studies independently or alongside other electrochemical techniques such as CV. ...
Phthalocyanines are aromatic or macrocyclic organic compounds and attract great attention due to their numerous properties. They have many high-tech applications in different areas of the industry such as dyestuffs, thermal printing screens, photovoltaic solar cells, membrane catalytic reactors, semiconductor materials and gas sensors. In the last decade, electrochemical sensor studies have accelerated with the catalytic lighting. It plays a dominant role in the development and implementation of new generation sensors. The aim of this study is to review the electrochemical methods based on electrode modification with phthalocyanines and to shed light on new application areas of phthalocyanines. The focal point was based on the sensor applications of phthalocyanines in the determination of drugs, pesticides, organic materials and metals etc. by electrochemical methods. Experimental conditions and some validation parameters of the sensor applications such as metal phthalocyanine types, indicator electrodes, selectivity, working ranges, detection limits, and analytical applications were discussed. Consequently, this is the first review dealing with the applications of phthalocyanines in electrochemical sensors for the sensitive determination of analytes in a variety of matrices.
... However, as shown in Figure 2f-h, the characteristic peak times, including t 1/2 , t rise and t fall , of male group are 104 %, 69 % and 109 % significantly higher than respective those of female groups (p < 0.0001 for each respective comparison). Since t 1/2 indicates the duration of exocytosis, and t rise and t fall represent the time of the opening and closing of fusion pore, [9] respectively, these results suggest the exocytotic fusion pore formed in male group stays longer than female group. With the same size of fusion pore, the longer duration results in more neurotransmitter released during exocytosis in male group ((2.04 � 0.15) × 10 6 , mean � SEM, n = 27) than female group ((1.68 � 0.09) × 10 6 , n = 28) compared with Mann-Whitney test (p < 0.05) (Figure 2i). ...
... The normalized d, schemed in Figure S4a, was calculated with Equation S2 to define whether the spike is well fitted with single-exponential or double-exponential function (see Note S1). [7a, 9,11] Since there is no standard criteria value for d to distinguish single-and double-exponential fitting, the spikes with d � 0.5 were considered more suitable for double-exponential fitting as reported before, [11] which corresponds to partial release. As shown in Figure S4b and Figure 2m, the percentage of spikes with d � 0.5 in male group (52.5 %) is significantly lower than that of female group (58.2 %) compared with Chi-Square test. ...
Quantitative measurements of sex difference in vesicle chemistry (i.e., chemical storage and release) at the single‐vesicle level are essential to understand sex differences in cognitive behaviors; however, such measurements are very challenging to conventional analytical methods. By using single‐vesicle electrochemistry, we find the duration of single exocytotic events of chromaffin cells prepared from male rats is statistically longer than that from female rats, leading to more neurotransmitter released in the male group. Further analysis reveals that a higher percentage of vesicles in the female group release part of the neurotransmitter, i.e., partial release, during exocytosis than that in male group. This sex dimorphism in neurotransmitter release in exocytosis might relate to the sex difference in the expression of voltage‐dependent calcium channels and membrane lipid composition. Our finding offers the first experimental evidence that sex dimorphism even exists in vesicle chemistry, providing a brand new viewpoint for understanding the sex dimorphism in exocytosis.
... Individual amperometric spikes were analyzed using the Quanta Analysis 8.20 program running under Igor Pro software (Wavemetrics, Portland, OR, USA) [32], allowing peak detection and integration plus the calculation of kinetic parameters. The threshold to detect spikes was set at twice the standard deviation of the noise. ...
The hypersecretory phenotype of adrenal chromaffin cells (CCs) from early spontaneously hypertensive rats (SHRs) mainly results from enhanced Ca2+-induced Ca2+-release (CICR). A key question is if these abnormalities can be traced to the prehypertensive stage. Spontaneous and stimulus-induced catecholamine exocytosis, intracellular Ca2+ signals, and dense-core granule size and density were examined in CCs from prehypertensive and hypertensive SHRs and compared with age-matched Wistar-Kyoto rats (WKY). During the prehypertensive stage, the depolarization-elicited catecholamine exocytosis was ~ 2.9-fold greater in SHR than in WKY CCs. Interestingly, in half of CCs the exocytosis was indistinguishable from WKY CCs, while it was between 3- and sixfold larger in the other half. Likewise, caffeine-induced exocytosis was ~ twofold larger in prehypertensive SHR. Accordingly, depolarization and caffeine application elicited [Ca2+]i rises ~ 1.5-fold larger in prehypertensive SHR than in WKY CCs. Ryanodine reduced the depolarization-induced secretion in prehypertensive SHR by 57%, compared to 14% in WKY CCs, suggesting a greater contribution of intracellular Ca2+ release to exocytosis. In SHR CCs, the mean spike amplitude and charge per spike were significantly larger than in WKY CCs, regardless of age and stimulus type. This difference in granule content could explain in part the enhanced exocytosis in SHR CCs. However, electron microscopy did not reveal significant differences in granule size between SHRs and WKY rats’ adrenal medulla. Nonetheless, preSHR and hypSHR display 63% and 82% more granules than WKY, which could explain in part the enhanced catecholamine secretion. The mechanism responsible for the heterogeneous population of prehypertensive SHR CCs and the bias towards secreting more medium and large granules remains unexplained.
