Dynamic Visualization and Quantification of Single Vesicle Opening and Content by Coupling Vesicle Impact Electrochemical Cytometry with Confocal Microscopy.

In this work, we introduce a novel method for visualization and quantitative measurement of the vesicle opening process by correlation of vesicle impact electrochemical cytometry (VIEC) with confocal microscopy. We have used a fluorophore conjugated to lipids to label the vesicle membrane and manipulate the membrane properties, which appears to make the membrane more susceptible to electroporation. The neurotransmitters inside the vesicles were visualized by use of a fluorescence false neurotransmitter 511 (FFN 511) through accumulation inside the vesicle via the neuronal vesicular monoamine transporter 2 (VMAT 2). Optical and electrochemical measurements of single vesicle electroporation were carried out using an in-house, disk-shaped, gold-modified ITO (Au/ITO) microelectrode device (5 nm thick, 33 µm diameter), which simultaneously acted as an electrode surface for VIEC and an optically transparent surface for confocal microscopy. As a result, the processes of adsorption, electroporation, and opening of single vesicles followed by neurotransmitter release on the Au/ITO surface have been simultaneously visualized and measured. Three opening patterns of single isolated vesicles were frequently observed. Comparing the vesicle opening patterns with their corresponding VIEC spikes, we propose that the behavior of the vesicular membrane on the electrode surface, including the adsorption time, residence time before vesicle opening, and the retention time after vesicle opening, are closely related to the vesicle content and size. Large vesicles with high content tend to adsorb to the electrode faster with higher frequency, followed by a shorter residence time before releasing their content, and their membrane remains on the electrode surface longer compared to the small vesicles with low content. With this approach, we start to unravel the vesicle opening process and to examine the fundamentals of exocytosis, supporting the proposed mechanism of partial or subquantal release in exocytosis.

Direct Measurement of Total Vesicular Catecholamine Content with Electrochemical Microwell Arrays.

We have designed and fabricated a microwell array chip (MWAC) to trap and detect the entire content of individual vesicles after disruption of the vesicular membrane by an applied electrical potential. To understand the mechanism of vesicle impact electrochemical cytometry (VIEC) in microwells, we simulated the rupture of the vesicles and subsequent diffusion of entrapped analytes. Two possibilities were tested: (i) the vesicle opens toward the electrode, and (ii) the vesicle opens away from the electrode. These two possibilities were simulated in the different microwells with varied depth and width. Experimental VIEC measurements of the number of molecules for each vesicle in the MWAC were compared to VIEC on a gold microdisk electrode as a control, and the quantified catecholamines between these two techniques was the same. We observed a prespike foot in a significant number of events (∼20%) and argue this supports the hypothesis that the vesicles rupture toward the electrode surface with a more complex mechanism including the formation of a stable pore intermediate. This study not only confirms that in standard VIEC experiments the whole content of the vesicle is oxidized and quantified at the surface of the microdisk electrode but actively verifies that the adsorbed vesicle on the surface of the electrode forms a pore in the vicinity of the electrode rather than away from it. The fabricated MWAC promotes our ability to quantify the content of vesicles accurately, which is fundamentally important in bioanalysis of the vesicles.

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.

Spatial Lipidomics Reveals Region and Long Chain Base Specific Accumulations of Monosialogangliosides in Amyloid Plaques in Familial Alzheimer's Disease Mice (5xFAD) Brain.

Ganglioside metabolism is significantly altered in Alzheimer's disease (AD), which is a progressive neurodegenerative disease prominently characterized by one of its pathological hallmarks, amyloid deposits or "senile plaques". While the plaques mainly consist of aggregated variants of amyloid-ß protein (Aß), recent studies have revealed a number of lipid species including gangliosides in amyloid plaques along with Aß peptides. It has been widely suggested that long chain (sphingosine) base (LCBs), C18:1-LCB and C20:1-LCB, containing gangliosides might play different roles in neuronal function in vivo. In order to elucidate region-specific aspects of amyloid-plaque associated C18:1-LCB and C20:1-LCB ganglioside accumulations, high spatial resolution (10 µm per pixel) matrix assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) of gangliosides in amyloid plaques was performed in hippocampal and adjacent cortical regions of 12 month old 5xFAD mouse coronal brain sections from two different stereotaxic coordinates (bregma points, -2.2 and -2.7 mm). MALDI-IMS uncovered brain-region (2 and 3D) and/or LCB specific accumulations of monosialogangliosides (GMs): GM1, GM2, and GM3 in the hippocampal and cortical amyloid plaques. The results reveal monosialogangliosides to be an important component of amyloid plaques and the accumulation of different gangliosides is region and LCB specific in 12 month old 5xFAD mouse brain. This is discussed in relation to amyloid-associated AD pathogenesis such as lipid related immune changes in amyloid plaques, AD specific ganglioside metabolism, and, notably, AD-associated impaired neurogenesis in the subgranular zone.

Dopamine Release Dynamics in the Tuberoinfundibular Dopamine System.

The relationship between neuronal impulse activity and neurotransmitter release remains elusive. This issue is especially poorly understood in the neuroendocrine system, with its particular demands on periodically voluminous release of neurohormones at the interface of axon terminals and vasculature. A shortage of techniques with sufficient temporal resolution has hindered real-time monitoring of the secretion of the peptides that dominate among the neurohormones. The lactotropic axis provides an important exception in neurochemical identity, however, as pituitary prolactin secretion is primarily under monoaminergic control, via tuberoinfundibular dopamine (TIDA) neurons projecting to the median eminence (ME). Here, we combined electrical or optogenetic stimulation and fast-scan cyclic voltammetry to address dopamine release dynamics in the male mouse TIDA system. Imposing different discharge frequencies during brief (3 s) stimulation of TIDA terminals in the ME revealed that dopamine output is maximal at 10 Hz, which was found to parallel the TIDA neuron action potential frequency distribution during phasic discharge. Over more sustained stimulation periods (150 s), maximal output occurred at 5 Hz, similar to the average action potential firing frequency of tonically active TIDA neurons. Application of the dopamine transporter blocker, methylphenidate, significantly increased dopamine levels in the ME, supporting a functional role of the transporter at the neurons' terminals. Lastly, TIDA neuron stimulation at the cell body yielded perisomatic release of dopamine, which may contribute to an ultrafast negative feedback mechanism to constrain TIDA electrical activity. Together, these data shed light on how spiking patterns in the neuroendocrine system translate to vesicular release toward the pituitary and identify how dopamine dynamics are controlled in the TIDA system at different cellular compartments.SIGNIFICANCE STATEMENT A central question in neuroscience is the complex relationship between neuronal discharge activity and transmitter release. By combining optogenetic stimulation and voltammetry, we address this issue in dopamine neurons of the neuroendocrine system, which faces particular spatiotemporal demands on exocytotic release; large amounts of neurohormone need to be secreted into the portal capillaries with precise timing to adapt to physiological requirements. Our data show that release is maximal around the neurons' default firing frequency. We further provide support for functional dopamine transport at the neurovascular terminals, shedding light on a long-standing controversy about the existence of neuroendocrine transmitter reuptake. Finally, we show that dopamine release occurs also at the somatodendritic level, providing a substrate for an ultrashort autoregulatory feedback loop.

Combined Amperometry and Electrochemical Cytometry Reveal Differential Effects of Cocaine and Methylphenidate on Exocytosis and the Fraction of Chemical Release.

Amperometry with nanotip electrodes has been applied to show cocaine and methylphenidate not only trigger declines in vesicle content and exocytotic catecholamine release in a model cell line but also differentially change the fraction of transmitter released from each individual vesicle. In addition, cocaine accelerates exocytotic release dynamics while they remain unchanged after methylphenidate treatment. The parameters from pre-spike feet for the two drugs are also in opposition, suggesting this aspect of release is affected differentially. As cocaine and methylphenidate are psychostimulants with similar pharmacologic action but have opposite effects on cognition, these results might provide a missing link between the regulation of exocytosis and vesicles and the effect of this regulation on cognition, learning, and memory. A speculative chemical mechanism of the effect of these drugs on vesicle content and exocytosis is presented.

On-Tissue Chemical Derivatization of Catecholamines Using 4-( N-Methyl)pyridinium Boronic Acid for ToF-SIMS and LDI-ToF Mass Spectrometry Imaging.

The analysis of small polar compounds with ToF-SIMS and MALDI-ToF-MS have been generally hindered by low detection sensitivity, poor ionization efficiency, ion suppression, analyte in-source fragmentation, and background spectral interferences from either a MALDI matrix and/or endogenous tissue components. Chemical derivatization has been a well-established strategy for improved mass spectrometric detection of many small molecular weight endogenous compounds in tissues. Here, we present a devised strategy to selectively derivatize and sensitively detect catecholamines with both secondary ion ejection and laser desorption ionization strategies, which are used in many imaging mass spectrometry (IMS) experiments. Chemical derivatization of catecholamines was performed by a reaction with a synthesized permanent pyridinium-cation-containing boronic acid molecule, 4-( N-methyl)pyridinium boronic acid, through boronate ester formation (boronic acid-diol reaction). The derivatization facilitates their sensitive detection with ToF-SIMS and LDI-ToF mass spectrometric techniques. 4-( N-Methyl)pyridinium boronic acid worked as a reactive matrix for catecholamines with LDI and improved the sensitivity of detection for both SIMS and LDI, while the isotopic abundances of the boron atom reflect a unique isotopic pattern for derivatized catecholamines in MS analysis. Finally, the devised strategy was applied, as a proof of concept, for on-tissue chemical derivatization and GCIB-ToF-SIMS (down to 3 µm per pixel spatial resolution) and LDI-ToF mass spectrometry imaging of dopamine, epinephrine, and norepinephrine in porcine adrenal gland tissue sections. MS/MS using collision-induced dissociation (CID)-ToF-ToF-SIMS was subsequently employed on the same tissue sections after SIMS and LDI mass spectrometry imaging experiments, which provided tandem MS information for the validation of the derivatized catecholamines in situ. This methodology can be a powerful approach for the selective and sensitive ionization/detection and spatial localization of diol-containing molecules such as aminols, vic-diols, saccharides, and glycans along with catecholamines in tissue sections with both SIMS and LDI/MALDI-MS techniques.

Electrochemical quantification of transmitter concentration in single nanoscale vesicles isolated from PC12 cells.

We use an electrochemical platform, nanoparticle tracking analysis, and differential centrifugation of single catecholamine vesicles to study the properties of nanometer transmitter vesicles, including the number of molecules, size, and catecholamine concentration inside. Vesicle impact electrochemical cytometry (VIEC) was used to quantify the catecholamine content of single vesicles in different batches isolated from pheochromocytoma (PC12) cells with different ultracentrifugation speeds. We show that, vesicles containing less catecholamine are obtained at subsequent centrifugation steps with higher speed (force). Important to quantification, the cumulative content after subsequent centrifugation steps is equivalent to that of one-step centrifugation at the highest speed, 70 000g. Moreover, as we count molecules in the vesicles, we compared molecular numbers from VIEC, flow VIEC, and intracellular VIEC to corresponding vesicle size measured by nanoparticle tracking analysis to evaluate catecholamine concentration in vesicles. The data suggest that vesicular catecholamine concentration is relatively constant and independent of the vesicular size, indicating vesicular transmitter content as a main factor regulating the vesicle size.

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis.

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity and the amount of neurotransmitter released from vesicles in single exocytosis events. It has been suggested that the reduction in neurotransmitters expelled is due to alterations in membrane biophysical properties when cells shrink in response to osmotic stress and with assumptions made that secretory vesicles in the cell cytoplasm are not affected by extracellular osmotic stress. Amperometry recording of exocytosis monitors what is released from cells the moment a vesicle fuses with the plasma membrane, but does not provide information on the vesicle content before the vesicle fusion is triggered. Therefore, by combining amperometry recording with other complementary analytical methods that are capable of characterizing the secretory vesicles before exocytosis at cells is triggered offers a broader overview for examining how secretory vesicles and the exocytosis process are affected by osmotic shock. We here describe how complementing amperometry recording with intracellular electrochemical cytometry and transmission electron microscopy (TEM) imaging can be used to characterize alterations in secretory vesicles size and neurotransmitter content at chromaffin cells in relation to exocytosis activity before and after exposure to osmotic stress. By linking the quantitative information gained from experiments using all three analytical methods, conclusions were previously made that secretory vesicles respond to extracellular osmotic stress by shrinking in size and reducing the vesicle quantal size to maintain a constant vesicle neurotransmitter concentration. Hence, this gives some clarification regarding why vesicles, in response to osmotic stress, reduce the amount neurotransmitters released during exocytosis release. The amperometric recordings here indicate this is a reversible process and that vesicle after osmotic shock are refilled with neurotransmitters when placed cells are reverted into an isotonic environment.

Nanopore Opening at Flat and Nanotip Conical Electrodes during Vesicle Impact Electrochemical Cytometry.

The oxidation of catecholamine at a microelectrode, following its release from individual vesicles, allows interrogation of the content of single nanometer vesicles with vesicle impact electrochemical cytometry (VIEC). Previous to this development, there were no methods available to quantify the chemical load of single vesicles. However, accurate quantification of the content is hampered by uncertainty in the proportion of substituent molecules reaching the electrode surface (collection efficiency). In this work, we use quantitative modeling to calculate this collection efficiency. For all vesicles except those at the very edge of the electrode, modeling shows that ∼100% oxidation efficiency is achieved when employing a 33 µm diameter disk microelectrode for VIEC, independent of the location of the vesicle release pore. We use this to experimentally determine a precise distribution of catecholamine in individual vesicles extracted from PC12 cells. In contrast, we calculate that when a nanotip conical electrode (∼4 µm length, ∼1.5 µm diameter at the base) is employed, as in intracellular VIEC (IVIEC), the current-time response depends strongly on the position of the catecholamine-releasing pore in the vesicle membrane. When vesicle release occurs with the pore opening occurring far from the electrode, lower currents and partial oxidation (∼75%) of the catecholamine are predicted, as compared to higher currents and ∼100% oxidation, when the pore is close to/at the electrode surface. As close agreement is observed between the experimentally measured vesicular content in intracellular and extracted vesicles from the same cell line using nanotip and disk electrodes, respectively, we conclude that pores open at the electrode surface. Not only does this suggest that electroporation of the vesicle membrane is the primary driving force for catecholamine release from vesicles at polarized electrodes, but it also indicates that IVIEC with nanotip electrodes can directly assess vesicular content without correction.

Electrochemical Investigation of the Interaction between Catecholamines and ATP.

The study of the colligative properties of adenosine 5'-triphosphate (ATP) and catecholamines has received the attention of scientists for decades, as they could explain the capabilities of secretory vesicles (SVs) to accumulate neurotransmitters. In this Article, we have applied electrochemical methods to detect such interactions in vitro, at the acidic pH of SVs (pH 5.5) and examined the effect of compounds having structural similarities that correlate with functional groups of ATP (adenosine, phosphoric acid and sodium phosphate salts) and catecholamines (catechol). Chronoamperometry and fast scan cyclic voltammetry (FSCV) provide evidence compatible with an interaction of the catechol and adenine rings. This interaction is also reinforced by an electrostatic interaction between the phosphate group of ATP and the protonated ammonium group of catecholamines. Furthermore, chronoamperometry data suggest that the presence of ATP subtlety reduces the apparent diffusion coefficient of epinephrine in aqueous media that adds an additional factor leading to a slower rate of catecholamine exocytosis. This adds another plausible mechanism to regulate individual exocytosis events to alter communication.

Vesicle impact electrochemical cytometry compared to amperometric exocytosis measurements.

Three new tools are discussed for understanding chemical communication between cells and primarily to delve into the content and structure of nanometer transmitter vesicles. These are amperometric measurements of exocytosis, vesicle impact electrochemical cytometry, and intracellular vesicle impact electrochemical cytometry. These are combining in the end nanoscale mass spectrometry imaging to begin determination of vesicle structure. These methods have provided solid evidence for the concept of open and closed exocytosis leading to partial release of the vesicle content during normal exocytosis. They have also been used to discover cases where the fraction of transmitter released is not changed, and other cases where the vesicle transmitter fraction released is altered, as with zinc, thought to alter cognition. Overall, the combination of these methods is showing us details of vesicular processes that would not be measureable without these micro and nano electrochemical methods.

Mechanistic Aspects of Vesicle Opening during Analysis with Vesicle Impact Electrochemical Cytometry.

Vesicle impact electrochemical cytometry (VIEC) has been used to quantify the vesicular transmitter content in mammalian vesicles. In the present study, we studied the mechanism of VIEC by quantifying the catecholamine content in single vesicles isolated from pheochromocytoma (PC12) cells. These vesicles contain about one tenth of the catecholamine compared with adrenal chromaffin vesicles. The existence of a prespike foot for many events suggests the formation of an initial transiently stable pore at the beginning of vesicle rupture. Increasing the detection temperature from 6 to 30 °C increases the possibility of vesicle rupture on the electrode, implying that there is a temperature-dependent process that facilitates electroporation. Natively larger vesicles are shown to rupture earlier and more frequently than smaller ones in VIEC. Likewise, manipulating vesicle content and size with drugs leads to similar trends. These data support the hypothesis that electroporation is the primary force for pore opening in VIEC. We further hypothesize that a critical step for initiating vesicle opening by electroporation is diffusion of membrane proteins away from the membrane region of contact with the electrode to allow closer contact, increasing the lateral potential field and thus facilitating electroporation.

DMSO Chemically Alters Cell Membranes to Slow Exocytosis and Increase the Fraction of Partial Transmitter Released.

Dimethyl sulfoxide (DMSO) is frequently used as a solvent in biological studies and as a vehicle for drug therapy; but the side effects of DMSO, especially on the cell environment, are not well understood, and controls with DMSO are not neutral at higher concentrations. Herein, electrochemical measurement techniques are applied to show that DMSO increases exocytotic neurotransmitter release, while leaving vesicular contents unchanged. In addition, the kinetics of release from DMSO-treated cells are faster than that of untreated ones. The results suggest that DMSO has a significant influence on the chemistry of the cell membrane, leading to alteration of exocytosis. A speculative chemical mechanism of the effect on the fusion pore during exocytosis is presented.

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

We report an approach to spatially resolve the content across nanometer neuroendocrine vesicles in nerve-like cells by correlating super high-resolution mass spectrometry imaging, NanoSIMS, with transmission electron microscopy (TEM). Furthermore, intracellular electrochemical cytometry at nanotip electrodes is used to count the number of molecules in individual vesicles to compare to imaged amounts in vesicles. Correlation between the NanoSIMS and TEM provides nanometer resolution of the inner structure of these organelles. Moreover, correlation with electrochemical methods provides a means to quantify and relate vesicle neurotransmitter content and release, which is used to explain the slow transfer of dopamine between vesicular compartments. These nanoanalytical tools reveal that dopamine loading/unloading between vesicular compartments, dense core and halo solution, is a kinetically limited process. The combination of NanoSIMS and TEM has been used to show the distribution profile of newly synthesized dopamine across individual vesicles. Our findings suggest that the vesicle inner morphology might regulate the neurotransmitter release event during open and closed exocytosis from dense core vesicles with hours of equilibrium needed to move significant amounts of catecholamine from the protein dense core despite its nanometer size.

Extracellular Osmotic Stress Reduces the Vesicle Size while Keeping a Constant Neurotransmitter Concentration.

Secretory cells respond to hypertonic stress by cell shrinking, which causes a reduction in exocytosis activity and the amount of signaling molecules released from single exocytosis events. These changes in exocytosis have been suggested to result from alterations in biophysical properties of cell cytoplasm and plasma membrane, based on the assumption that osmotic stress does not affect the secretory vesicle content and size prior to exocytosis. To further investigate whether vesicles in secretory cells are affected by the osmolality of the extracellular environment, we used intracellular electrochemical cytometry together with transmission electron microscopy imaging to quantify and determine the catecholamine concentration of dense core vesicles in situ before and after cell exposure to osmotic stress. In addition, single cell amperometry recordings of exocytosis at chromaffin cells were used to monitor the effect on exocytosis activity and quantal release when cells were exposed to osmotic stress. Here we show that hypertonic stress hampers exocytosis secretion after the first pool of readily releasable vesicles have been fused and that extracellular osmotic stress causes catecholamine filled vesicles to shrink, mainly by reducing the volume of the halo solution surrounding the protein matrix in dense core vesicles. In addition, the vesicles demonstrate the ability to perform adjustments in neurotransmitter content during shrinking, and intracellular amperometry measurements in situ suggest that vesicles reduce the catecholamine content to maintain a constant concentration within the vesicle compartment. Hence, the secretory vesicles in the cell cytoplasm are highly affected and respond to extracellular osmotic stress, which gives a new perspective to the cause of reduction in quantal size by these vesicles when undergoing exocytosis.

Excited Fluorophores Enhance the Opening of Vesicles at Electrode Surfaces in Vesicle Electrochemical Cytometry.

Electrochemical cytometry is a method developed recently to determine the content of an individual cell vesicle. The mechanism of vesicle rupture at the electrode surface involves the formation of a pore at the interface between a vesicle and the electrode through electroporation, which leads to the release and oxidation of the vesicle's chemical cargo. We have manipulated the membrane properties using excited fluorophores conjugated to lipids, which appears to make the membrane more susceptible to electroporation. We propose that by having excited fluorophores in close contact with the membrane, membrane lipids (and perhaps proteins) are oxidized upon production of reactive oxygen species, which then leads to changes in membrane properties and the formation of water defects. This is supported by experiments in which the fluorophores were placed on the lipid tail instead of the headgroup, which leads to a more rapid onset of vesicle opening. Additionally, application of DMSO to the vesicles, which increases the membrane area per lipid, and decreasing the membrane thickness result in the same enhancement in vesicle opening, which confirms the mechanism of vesicle opening with excited fluorophores in the membrane. Light-induced manipulation of membrane vesicle pore opening might be an attractive means of controlling cell activity and exocytosis. Additionally, our data confirm that in experiments in which cells or vesicle membranes are labeled for fluorescence monitoring, the properties of the excited membrane change substantially.

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.

Cholesterol Alters the Dynamics of Release in Protein Independent Cell Models for Exocytosis.

Neurons communicate via an essential process called exocytosis. Cholesterol, an abundant lipid in both secretory vesicles and cell plasma membrane can affect this process. In this study, amperometric recordings of vesicular dopamine release from two different artificial cell models created from a giant unilamellar liposome and a bleb cell plasma membrane, show that with higher membrane cholesterol the kinetics for vesicular release are decelerated in a concentration dependent manner. This reduction in exocytotic speed was consistent for two observed modes of exocytosis, full and partial release. Partial release events, which only occurred in the bleb cell model due to the higher tension in the system, exhibited amperometric spikes with three distinct shapes. In addition to the classic transient, some spikes displayed a current ramp or plateau following the maximum peak current. These post spike features represent neurotransmitter release from a dilated pore before constriction and show that enhancing membrane rigidity via cholesterol adds resistance to a dilated pore to re-close. This implies that the cholesterol dependent biophysical properties of the membrane directly affect the exocytosis kinetics and that membrane tension along with membrane rigidity can influence the fusion pore dynamics and stabilization which is central to regulation of neurochemical release.