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Both, patch amperometry (PA) and intracellular patch electrochemistry (IPE) take advantage of a recording configuration where an electrochemical detector – carbon fiber electrode (CFE) – is housed inside a patch pipette. PA, which is employed in cell-attached or excised inside-out patch clamp configuration, offers high resolution patch capacitance measurements with simultaneous amperometric detection of catecholamines released during exocytosis. The method provides precise information on single vesicle size and quantal content, fusion pore conductance and permeability of the pore for catecholamines. IPE, on the other hand, measures cytosolic catecholamines that diffuse into the patch pipette following membrane rupture to achieve the whole-cell configuration. In amperometric mode, IPE detects total catechols, whereas in cyclic voltammetric mode it provides more specific information on the nature of the detected molecules and may selectively quantify catecholamines, providing a direct approach to determine cytosolic concentrations of catecholaminergic transmitters and their metabolites. Here we provide detailed instructions on setting up PA and IPE, performing experiments and analyzing the data.

Complementary metal-oxide-semiconductor (CMOS) microelectrode arrays integrate amplifier arrays with on-chip electrodes, offering high-throughput platforms for electrochemical sensing with high spatial and temporal resolution. Such devices have been developed for highly parallel constant voltage amperometric detection of transmitter release from multiple cells with single-vesicle resolution. Cyclic voltammetry (CV) is an electrochemical method that applies voltage waveforms, which provides additional information about electrode properties and about the nature of analytes. A 16-channel, 64-electrode-per-channel CMOS integrated circuit (IC) fabricated in a 0.5 μm CMOS process for CV is demonstrated. Each detector consists of only 11 transistors and an integration capacitor with a unit dimension of 0.0015 mm2. The device was postfabricated using Pt as the working electrode material with a shifted electrode design, which makes it possible to redefine the size and the location of working electrodes. The system incorporating cell-sized (8 μm radius) microelectrodes was validated with dopamine injection tests and CV measurements of potassium ferricyanide at a 1 V/s scanning rate. The cyclic voltammograms were in excellent agreement with theoretical predictions. The technology enables rigorous characterization of electrode performance for the application of CMOS microelectrode arrays in low-noise amperometric measurements of quantal transmitter release as well as other biosensing applications.

Neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease, are considered incurable and significantly reduce the quality of life of the patients. A variety of drugs that modulate neurotransmitter levels have been used for the treatment of the neurodegenerative diseases but with limited efficacy. In this work, an amperometric complementary metal‐oxide‐semiconductor (CMOS) chip is used for high‐throughput drug testing with respect to the modulation of transmitter release from single vesicles using chromaffin cells prepared from bovine adrenal glands as a model system. Single chromaffin cell amperometry was performed with high efficiency on the surface‐modified CMOS chip and follow‐up whole‐cell patch‐clamp experiments were performed to determine the readily releasable pool sizes. We show that the antidepressant drug bupropion significantly increases the amount of neurotransmitter released in individual quantal release events. The antidepressant drug citalopram accelerates rapid neurotransmitter release following stimulation and follow‐up patch‐clamp experiments reveal that this is because of the increase in the pool of readily releasable vesicles. These results shed light on the mechanisms by which bupropion and citalopram may be potentially effective in the treatment of neurodegenerative diseases. These results demonstrate that the CMOS amperometry chip technology is an excellent tool for drug testing to determine the specific mechanisms by which they modulate neurotransmitter release., Bupropion and citalopram are antidepressant drugs that have been used for decades. However, the effect of these two drugs on the release frequency, quantal size, and kinetics of neurotransmitter release events has not been studied before. In this work, we used a surface‐modified complementary metal‐oxide‐semiconductor integrated circuit sensor array to perform high‐throughput amperometry recordings of chromaffin cells treated with these two drugs. The results show that bupropion increases the quantal size of neurotransmitter release events and citalopram increases the release frequency by increasing the readily releasable pool sizes for chromaffin cells. The potential therapeutic applications of the two drugs on neurodegenerative diseases have been discussed.

Human neuroblastoma cells, SH-SY5Y, are often used as a neuronal model to study Parkinson's disease and dopamine release in the substantia nigra, a midbrain region that plays an important role in motor control. Using amperometric single-cell recordings of single vesicle release events, we can study molecular manipulations of dopamine release and gain a better understanding of the mechanisms of neurological diseases. However, single-cell analysis of neurotransmitter release using traditional techniques yields results with very low throughput. In this paper, we will discuss a monolithically-integrated CMOS sensor array that has the low-noise performance, fine temporal resolution, and 1024 parallel channels to observe dopamine release from many single cells with single-vesicle resolution. The measured noise levels of our transimpedance amplifier are 415, 622, and 1083 ${\text{fA}}_{{\text{RMS}}}$ , at sampling rates of 10, 20, and 30 kS/s, respectively, without additional filtering. Post-CMOS processing is used to monolithically integrate 1024 on-chip gold electrodes, with an individual electrode size of 15 μm × 15 μm, directly on 1024 transimpedance amplifiers in the CMOS device. SU-8 traps are fabricated on individual electrodes to allow single cells to be interrogated and to reject multicellular clumps. Dopamine secretions from 76 cells are simultaneously recorded by loading the CMOS device with SH-SY5Y cells. In the 42-s measurement, a total of 7147 single vesicle release events are monitored. The study shows the CMOS device's capability of recording vesicle secretion at a single-cell level, with 1024 parallel channels, to provide detailed information on the dynamics of dopamine release at a single-vesicle resolution.

Release of neurotransmitters from synaptic vesicles begins with a narrow fusion pore, the structure of which remains unresolved. To obtain a structural model of the fusion pore, we performed coarse-grained molecular dynamics simulations of fusion between a nanodisc and a planar bilayer bridged by four partially unzipped SNARE complexes. The simulations revealed that zipping of SNARE complexes pulls the polar C-terminal residues of the synaptobrevin 2 and syntaxin 1A transmembrane domains to form a hydrophilic core between the two distal leaflets, inducing fusion pore formation. The estimated conductances of these fusion pores are in good agreement with experimental values. Two SNARE protein mutants inhibiting fusion experimentally produced no fusion pore formation. In simulations in which the nanodisc was replaced by a 40-nm vesicle, an extended hemifusion diaphragm formed but a fusion pore did not, indicating that restricted SNARE mobility is required for rapid fusion pore formation. Accordingly, rapid fusion pore formation also occurred in the 40-nm vesicle system when SNARE mobility was restricted by external forces. Removal of the restriction is required for fusion pore expansion.

Fluorescence imaging is often used to monitor dynamic cellular functions under conditions of very low light intensities to avoid photodamage to the cell and rapid photobleaching. Determination of the time of a fluorescence change relative to a rapid high time-resolution event, such as an action potential or pulse stimulation, is challenged by the low photon rate and the need to use imaging frame durations that limit the time resolution. To overcome these limitations, we developed a time superresolution method named event correlation microscopy that aligns repetitive events with respect to the high time-resolution events. We describe the algorithm of the method, its step response function, and a theoretical, computational, and experimental analysis of its precision, providing guidelines for camera exposure time settings depending on imaging signal properties and camera parameters for optimal time resolution. We also demonstrate the utility of the method to recover rapid nonstepwise kinetics by deconvolution fits. The event correlation microscopy method provides time superresolution beyond the photon rate limit and imaging frame duration with well-defined precision.

SUMMARY Many neurotransmitters are organic ions that carry a net charge, and their release from secretory vesicles is therefore an electrodiffusion process. The selectivity of early exocytotic fusion pores is investigated by combining electrodiffusion theory, measurements of amperometric foot signals from chromaffin cells with anion substitution, and molecular dynamics simulation. The results reveal that very narrow fusion pores are cation selective, but more dilated fusion pores become anion permeable. The transition occurs around a fusion pore conductance of ~300 pS. The cation selectivity of a narrow fusion pore accelerates the release of positively charged transmitters such as dopamine, noradrenaline, adrenaline, serotonin, and acetylcholine, while glutamate release may require a more dilated fusion pore., Graphical Abstract, In brief For transmission, a fusion pore forms when vesicle and target membranes are brought together by SNARE proteins. Delacruz et al. demonstrate that selectivity of the pore accelerates release of positively charged transmitters such as dopamine, noradrenaline, adrenaline, serotonin, and acetylcholine, while glutamate release may require a more dilated fusion pore.

Neurotransmitter release occurs in the form of quantal events by fusion of secretory vesicles with the plasma membrane, and begins with the formation of a fusion pore that has a conductance similar to that of a large ion channel or gap junction. In this review, we propose mechanisms of fusion pore formation and discuss their implications for fusion pore structure and function. Accumulating evidence indicates a direct role of soluble N-ethylmaleimide-sensitive-factor attachment receptor proteins in the opening of fusion pores. Fusion pores are likely neither protein channels nor purely lipid, but are of proteolipidic composition. Future perspectives to gain better insight into the molecular structure of fusion pores are discussed.

The t-SNARE complex plays a central role in neuronal fusion. Its components, syntaxin-1 and SNAP25, are largely present in individual clusters and partially colocalize at the presumptive fusion site. How these protein clusters modify local lipid composition and membrane morphology is largely unknown. In this work, using coarse-grained molecular dynamics, the transmembrane domains (TMDs) of t-SNARE complexes are shown to form aggregates leading to formation of lipid nanodomains, which are enriched in cholesterol, phosphatidylinositol 4,5-bisphosphate, and gangliosidic lipids. These nano-domains induce membrane curvature that would promote a closer contact between vesicle and plasma membrane.

SNAP-25 is a Q-SNARE protein mediating exocytosis of neurosecretory vesicles including chromaffin granules. Previous results with a SNAP-25 construct lacking the nine C terminal residues (SNAP-25Δ9) showed changed fusion pore properties (Fang et al., 2008), suggesting a model for fusion pore mechanics that couple C terminal zipping of the SNARE complex to the opening of the fusion pore. The deleted fragment contains the positively charged residues R198 and K201, adjacent to layers 7 and 8 of the SNARE complex. To determine how fusion pore conductance and dynamics depend on these residues, single exocytotic events in bovine chromaffin cells expressing R198Q, R198E, K201Q, or K201E mutants were investigated by carbon fiber amperometry and cell-attached patch capacitance measurements. Coarse grain molecular dynamics simulations revealed spontaneous transitions between a loose and tightly zippered state at the SNARE complex C terminus. The SNAP-25 K201Q mutant showed no changes compared with SNAP-25 wild-type. However, K201E, R198Q, and R198E displayed reduced release frequencies, slower release kinetics, and prolonged fusion pore duration that were correlated with reduced probability to engage in the tightly zippered state. The results show that the positively charged amino acids at the SNAP-25 C terminus promote tight SNARE complex zippering and are required for high release frequency and rapid release in individual fusion events.

Amperometry is a powerful method to record quantal release events from chromaffin cells and is widely used to assess how specific drugs modify quantal size, kinetics of release, and early fusion pore properties. Surface-modified CMOS-based electrochemical sensor arrays allow simultaneous recordings from multiple cells. A reliable, low-cost technique is presented here for efficient targeting of single cells specifically to the electrode sites. An SU-8 microwell structure is patterned on the chip surface to provide insulation for the circuitry as well as cell trapping at the electrode sites. A shifted electrode design is also incorporated to increase the flexibility of the dimension and shape of the microwells. The sensitivity of the electrodes is validated by a dopamine injection experiment. Microwells with dimensions slightly larger than the cells to be trapped ensure excellent single-cell targeting efficiency, increasing the reliability and efficiency for on-chip single-cell amperometry measurements. The surface-modified device was validated with parallel recordings of live chromaffin cells trapped in the microwells. Rapid amperometric spikes with no diffusional broadening were observed, indicating that the trapped and recorded cells were in very close contact with the electrodes. The live cell recording confirms in a single experiment that spike parameters vary significantly from cell to cell but the large number of cells recorded simultaneously provides the statistical significance.

Although several proteins have been implicated in secretory vesicle tethering, the identity and mechanical properties of the components forming the physical vesicle-plasma membrane link remain unknown. Here we present the first experimental measurements of nanomechanical properties of secretory vesicle-plasma membrane tethers using combined AFM force clamp and TIRF microscopy on membrane sheets from PC12 cells expressing the vesicle marker ANF-eGFP. Application of pulling forces generated tether extensions composed of multiple steps with variable length. The frequency of short (

The SNARE complex consists of the three proteins synaptobrevin-2, syntaxin, and synaptosomal-associated protein 25 (SNAP25) and is thought to execute a large conformational change as it drives membrane fusion and exocytosis. The relation between changes in the SNARE complex and fusion pore opening is, however, still unknown. We report here a direct measurement relating a change in the SNARE complex to vesicle fusion on the millisecond time scale. In individual chromaffin cells, we tracked conformational changes in SNAP25 by total internal reflection fluorescence resonance energy transfer (FRET) microscopy while exocytotic catecholamine release from single vesicles was simultaneously recorded using a microfabricated electrochemical detector array. A local rapid and transient FRET change occurred precisely where individual vesicles released catecholamine. To overcome the low time resolution of the imaging frames needed to collect sufficient signal intensity, a method named event correlation microscopy was developed, which revealed that the FRET change was abrupt and preceded the opening of an exocytotic fusion pore by ∼90 ms. The FRET change correlated temporally with the opening of the fusion pore and not with its dilation.

Vesicle fusion is mediated by an assembly of SNARE proteins between opposing membranes, but it is unknown whether transmembrane domains (TMDs) of SNARE proteins serve mechanistic functions that go beyond passive anchoring of the force-generating SNAREpin to the fusing membranes. Here, we show that conformational flexibility of synaptobrevin-2 TMD is essential for efficient Ca2+-triggered exocytosis and actively promotes membrane fusion as well as fusion pore expansion. Specifically, the introduction of helix-stabilizing leucine residues within the TMD region spanning the vesicle’s outer leaflet strongly impairs exocytosis and decelerates fusion pore dilation. In contrast, increasing the number of helix-destabilizing, ß-branched valine or isoleucine residues within the TMD restores normal secretion but accelerates fusion pore expansion beyond the rate found for the wildtype protein. These observations provide evidence that the synaptobrevin-2 TMD catalyzes the fusion process by its structural flexibility, actively setting the pace of fusion pore expansion. DOI: http://dx.doi.org/10.7554/eLife.17571.001, eLife digest Neurons signal to other cells by releasing chemicals known as neurotransmitters. The neurotransmitters are stored in the neuron in small membrane-bound compartments called vesicles. When a neuron receives an electrical impulse, this ultimately triggers the vesicles to fuse with the cell membrane and release their contents into the gap between the neurons. This process is known as exocytosis. Other cells called neuroendocrine cells, which can receive signals from neurons, also undergo exocytosis to release chemicals into the bloodstream. A group of membrane-bound proteins called SNAREs help a vesicle to fuse with the cell membrane. SNARE proteins are embedded in both the vesicle and cell membrane, and force them into close proximity. When the two membranes make contact, a small channel called the fusion pore forms and expands to release the vesicle’s contents out of the cell. Synaptobrevin-2 is a SNARE protein found in the vesicle membrane. The part of the protein that sits in the membrane is called the transmembrane domain; however, it is not clear whether this domain plays any role in membrane fusion. The transmembrane domain of synaptobrevin-2 is rich in certain amino acids that are thought to make it flexible, thereby allowing it to bend and tilt in the membrane. Dhara, Yarzagaray et al. altered these amino acids in such a way that made this domain either more or less flexible than in the normal protein. The results show that in both neurons and a type of neuroendocrine cell called chromaffin cells, exocytosis is significantly reduced and the fusion pores open more slowly when the transmembrane domain is less flexible. By contrast, exocytosis occurs normally when the transmembrane domain is more flexible; however, the fusion pore expands more rapidly than normal. These results suggest that the flexibility of the transmembrane domain of synaptobrevin-2 promotes membrane fusion and sets the pace at which the fusion pore expands. It is likely that the transmembrane domain disturbs the surrounding membrane in a way that enables these events to happen. Further work is needed to address whether this is the case. DOI: http://dx.doi.org/10.7554/eLife.17571.002

Prostaglandins inhibit insulin secretion in a manner similar to that of norepinephrine (NE) and somatostatin. As NE inhibits endocytosis as well as exocytosis, we have now examined the modulation of endocytosis by prostaglandin E1(PGE1). Endocytosis following exocytosis was recorded by whole-cell patch clamp capacitance measurements in INS-832/13 cells. Prolonged depolarizing pulses producing a high level of Ca2+influx were used to stimulate maximal exocytosis and to deplete the readily releasable pool (RRP) of granules. This high Ca2+influx eliminates the inhibitory effect of PGE1on exocytosis and allows specific characterization of the inhibitory effect of PGE1on the subsequent compensatory endocytosis. After stimulating exocytosis, endocytosis was apparent under control conditions but was inhibited by PGE1in a Pertussis toxin-sensitive (PTX)-insensitive manner. Dialyzing a synthetic peptide mimicking the C-terminus of the α-subunit of the heterotrimeric G-protein Gzinto the cells blocked the inhibition of endocytosis by PGE1, whereas a control-randomized peptide was without effect. These results demonstrate that PGE1inhibits endocytosis and Gzmediates the inhibition.

During exocytosis, fusion pores form the first aqueous connection that allows escape of neurotransmitters and hormones from secretory vesicles. Although it is well established that SNARE proteins catalyze fusion, the structure and composition of fusion pores remain unknown. Here, we exploited the rigid framework and defined size of nanodiscs to interrogate the properties of reconstituted fusion pores, using the neurotransmitter glutamate as a content-mixing marker. Efficient Ca(2+)-stimulated bilayer fusion, and glutamate release, occurred with approximately two molecules of mouse synaptobrevin 2 reconstituted into ∼6-nm nanodiscs. The transmembrane domains of SNARE proteins assumed distinct roles in lipid mixing versus content release and were exposed to polar solvent during fusion. Additionally, tryptophan substitutions at specific positions in these transmembrane domains decreased glutamate flux. Together, these findings indicate that the fusion pore is a hybrid structure composed of both lipids and proteins.

The role of Ca2+in synaptic vesicle endocytosis remains uncertain due to the diversity in various preparations where several forms of endocytosis may contribute variably in different conditions. Although recent studies have demonstrated that Ca2+is important for clathrin-mediated endocytosis (CME), the mechanistic role of Ca2+in CME remains to be elucidated. By monitoring CME of single vesicles in mouse chromaffin cells with cell-attached capacitance measurements that offer millisecond time resolution, we demonstrate that the dynamics of vesicle fission during CME is Ca2+dependent but becomes Ca2+independent in synaptotagmin 1 (Syt1) knock-out cells. Our results thus suggest that Syt1 is necessary for the Ca2+dependence of CME.

The advent of organic electronics has made available a host of materials and devices with unique properties for interfacing with biology.1–2 One example is the use of conducting polymer coatings on metal electrodes that are implanted in the central nervous system and interface electrically with neurons, providing stimulation and recording the neuron's electrical activity.3–5 Coating a metal electrode with a conducting polymer has been shown to lower the electrical impedance and decrease the mechanical properties mismatch at the interface with tissue, with beneficial effects on the lifetime of the implant.3, 6 Conducting polymers can also be functionalized with biomolecules that stimulate neural growth and minimize the immune response to the implant.3–5, 7 Other examples are organic electronic ion pumps,8 and ion transistors,9 which are recently invented devices capable of precise delivery of neurotransmitters to neurons. These devices were recently implanted in the ear of a guinea pig and were shown to control its hearing.10 Conducting polymers such as poly(3,4-ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT:PSS), a material that has been shown to be biocompatible with a variety of different cells,1 have been used for these applications. These examples highlight the main advantages that organic electronic materials bring to the interface with biology, including their “soft” nature, which offers better mechanical compatibility with tissue than traditional electronic materials, and natural compatibility with mechanically flexible substrates, which paves the way for the development of implants that better conform to the non-planar shape of organs. Finally, the ability of organics to transport ionic in addition to electronic charge creates the opportunity to interface with electrically active cells in novel ways, as the work on ion pumps indicates.

The modulation of endocytosis following exocytosis by noradrenaline (NA), a physiological inhibitor of insulin secretion, was investigated in INS 832/13 cells using patch-clamp capacitance measurements. Endocytosis was inhibited by NA in a pertussis toxin-insensitive manner. Dialysing a synthetic peptide mimicking the C-terminus of the α-subunit of Gz into the cells blocked the inhibition of endocytosis by NA. Cell-attached capacitance measurements indicated that inhibition by NA was due to a decreased number of endocytotic events without a change in vesicle size. Analysis of fission pore closure kinetics revealed two distinct fission modes, with NA selectively inhibiting the rapid fission pore closure events. Comparison of the actions of NA and deltamethrin, a calcineurin antagonist and potent inhibitor of endocytosis, demonstrated that they inhibit endocytosis by different mechanisms. These findings establish novel actions for NA and Gz in insulin-secreting cells and possibly other cell types.

The molecular mechanisms responsible for the ‘distal’ effect by which noradrenaline (NA) blocks exocytosis in the β-cell were examined by whole-cell and cell-attached patch clamp capacitance measurements in INS 832/13 β-cells. NA inhibited Ca2+-evoked exocytosis by reducing the number of exocytotic events, without modifying vesicle size. Fusion pore properties also were unaffected. NA-induced inhibition of exocytosis was abolished by a high level of Ca2+ influx, by intracellular application of antibodies against the G protein subunit Gβ and was mimicked by the myristoylated βγ-binding/activating peptide mSIRK. NA-induced inhibition was also abolished by treatment with BoNT/A, which cleaves the C-terminal nine amino acids of SNAP-25, and also by a SNAP-25 C-terminal-blocking peptide containing the BoNT/A cleavage site. These data indicate that inhibition of exocytosis by NA is downstream of increased [Ca2+]i and is mediated by an interaction between Gβγ and the C-terminus of SNAP-25, as is the case for inhibition of neurotransmitter release. Remarkably, in the course of this work, a novel effect of NA was discovered. NA induced a marked retardation of the rate of refilling of the readily releasable pool (RRP) of secretory granules. This retardation was specifically abolished by a Gαi1/2 blocking peptide demonstrating that the effect is mediated via activation of Gαi1 and/or Gαi2.

The release of neurotransmitters and hormones from secretory vesicles plays a fundamental role in the function of the nervous system including neuronal communication. High-throughput testing of drugs modulating transmitter release is becoming an increasingly important area in the fields of cell biology, neurobiology, and neurology. Carbon-fiber amperometry provides high-resolution measurements of amount and time course of the transmitter release from single vesicles, and their modulation by drugs and molecular manipulations. However, these methods do not enable the rapid collection of data from a large number of cells. To allow this testing, we have developed a complementary metal-oxide semiconductor (CMOS) potentiostat circuit that can be scaled to a large array. In this paper, we present two post-CMOS fabrication methods to incorporate the electrochemical electrode material. We demonstrate by proof of principle the feasibility of on-chip electrochemical measurements of dopamine, and catecholamine release from adrenal chromaffin cells. The measurement noise is consistent with the typical electrode noise in recordings with external amplifiers. The electronic noise of the potentiostat in recordings with 400-?s integration time is ~ 0.11 pA and is negligible compared to the inherent electrode noise.

A 30- $\mu {\rm W}$ wireless fast-scan cyclic voltammetry monitoring integrated circuit for ultra-wideband (UWB) transmission of dopamine release events in freely-behaving small animals is presented. On-chip integration of analog background subtraction and UWB telemetry yields a 32-fold increase in resolution versus standard Nyquist-rate conversion alone, near a four-fold decrease in the volume of uplink data versus single-bit, third-order, delta-sigma modulation, and more than a 20-fold reduction in transmit power versus narrowband transmission for low data rates. The 1.5- ${\rm mm}^{2}$ chip, which was fabricated in 65-nm CMOS technology, consists of a low-noise potentiostat frontend, a two-step analog-to-digital converter (ADC), and an impulse-radio UWB transmitter (TX). The duty-cycled frontend and ADC/UWB-TX blocks draw 4 $\mu {\rm A}$ and 15 $\mu {\rm A}$ from 3-V and 1.2-V supplies, respectively. The chip achieves an input-referred current noise of 92 ${\rm pA}_{\rm rms}$ and an input current range of $\pm {430}~{\rm nA}$ at a conversion rate of 10 kHz. The packaged device operates from a 3-V coin-cell battery, measures 4.7 $\,\times\,$ 1.9 ${\rm cm}^{2}$ , weighs 4.3 g (including the battery and antenna), and can be carried by small animals. The system was validated by wirelessly recording flow-injection of dopamine with concentrations in the range of 250 nM to 1 $\mu {\rm M}$ with a carbon-fiber microelectrode (CFM) using 300-V/s FSCV.

Synaptic transmission and cell to cell communication in the human body are frequently characterized by the release of charged transmitters and other chemical mediators from secretory vesicles or granules which then impinge on specific receptor molecules expressed on target cells1,2,3. Depending on the excitable nature, the initiating cells respond to chemical inputs by releasing vesicular granules containing specific compounds or by inducing an electrical wave such as an action potential (AP). The process of vesicle fusion with the cell plasma membrane upon stimulation and subsequent release of the granular contents (i.e. in the form of quanta) into the extracellular environment is termed exocytosis4. When measured electrochemically such release events reveal a distinctive temporal response5,6. Exocytosis recordings are also often employed to characterize the mechanism of drug action on cells. For example, amperometric recordings have shown that the Parkinson’s drug L-Dopa increases the quantal size7, i.e. the total released charge increases, a consequence of increase vesicle size. There is thus a need to develop high throughput, scalable and multi-functional electronic instrumentation in order to characterize the action of various pharmacological inhibitors, toxins and stimulants on vesicle release. Transmitter and granular release can be specifically stimulated or inhibited depending on the cell type under study. In neurons, electrical excitations in the form of action potentials (AP) propagate along the axon and stimulate neurotransmitter release in the region between the axon terminus of the pre-synaptic neuron and the dendritic spine of the post-synaptic neuron [Fig. 1(a)] called the synapse. The released transmitters impinge on specific receptors on the post-synaptic neuron exciting or inhibiting action potential generation. In immune cells such as mast cells on the contrary, exocytosis can be induced through a receptor effector function where a specific antigen-receptor interaction causes a signal cascade within the cell, culminating in the release of chemical mediators which causes an allergic response. The released compounds from mast cells impinge on cells expressing specific receptors (such as the histamine receptor on smooth muscle cells) [Fig. 1(c)] and elicit a downstream response. In this study we seek to create a CMOS bio-sensor capable of detecting granule release from mast cells as a function of transmitter-receptor induced signaling. We then extend the approach to measuring depolarization induced activity from chromaffin cells where it can function as an electronic post-synaptic sensor [Fig. 1(d)]. Such a system not only provides a test bench for fundamental exocytotic analysis by monitoring release from vesicles and action potential’s with high temporal resolution, which is paramount in understanding cellular kinetics and establishing rapid screening procedures but also sets a promising route towards future artificial synapse systems and ionic-electronic interfacing circuitry. Figure 1 The cell-transistor synapse. The rat basophilic leukemia cell (RBL-2H3) is a tumor cell line used frequently as an experimental model for mucosal mast cells8. The release of inflammatory mediators from mast cells is the primary event in an allergic response9. These cells serve as a robust model for understanding the underlying biophysical and biochemical mechanism which couples signals originating at the membrane receptor with a biological effector function. Immunoglobulins of the IgE class serve as antigenic receptors which are anchored to cells via the membrane protein complex FceRI. Upon stimulation with multivalent antigen, the receptors crosslink causing a signal cascade within the cell, which eventually culminates in the secretion of preformed mediators stored in the cellular granules. Mast cells form a specialized niche of the immune system, because the triggered cellular activity is immediate. Depending on the particular type of mast cells or basophil’s, secretion occurs within seconds to minutes following the IgE cross linking step. Mast cells also provide a meaningful model for cell activation by an immunological stimulus, i.e., by an antibody-antigen reaction. We further demonstrate the device detection capability using chromaffin cells of the bovine adrenal medulla to detect neurotransmitter release and related electrical activity. The chromaffin cell is an excitable cell and allows us to study stimulus secretion coupling as mediated by both calcium entry and voltage gated channels, i.e., exocytosis induced by depolarization. Current methods of monitoring exocytosis include fluorescent techniques1,8,10,11, carbon-fiber amperometry12 and membrane capacitance measurements13. Fluorescence techniques often require labels and sophisticated optics, which increases the complexity of the experiment. On the other hand, amperometry may be prone to noise due to low current levels involved, relies on faradaic chemistry for detection, and is challenging to miniaturize in terms of pixel density, although recent efforts have resulted in significant improvements14,15,16. Non-faradaic transistor-based measurements on the contrary extend the detection capability to electrochemically inactive molecules, are extremely sensitive to surface adsorption, record cellular signals with a high degree of temporal sensitivity, present a naturally occurring high impedance node due to the gate oxide and can render sub-cellular spatial resolution17,18,19,20 with very low input referred noise characteristics21. Previous work on transistor-based cellular sensing has primarily focused on recording electrical activity from excitable cells such cardiac myocytes22,23 and nerve cells21,24. Recently Stern and co-workers25 extended transistor based sensing to detect antigen-stimulated T-cell activation detection by CMOS-compatible semiconducting nanowire sensors, however the exact signal generating mechanism was not investigated. In another report, transistor recording of vesicle release from chromaffin cells26 was demonstrated using open-gate ISFET’s. The recorded signal was attributed solely to the change in the local pH across the double-layer interface which leads to protonation of the surface and hence a change in surface potential. In this work, we extend this understanding and demonstrate the use of CMOS compatible floating gate transistors as cell based biosensors. Specifically we demonstrate that transistors can detect degranulation from electrically non-excitable cells such as the RBL-2H3 cell line and exocytosis from chromaffin cells. In addition to extracellular pH variation, we provide preliminary evidence suggesting direct molecular binding to the sensor surface as a signal generating mechanism. Since the nature of the FET interface allows one to record both electrochemical and ionic activities simultaneously we demonstrate the detection of AP’s and vesicle release simultaneously. Furthermore, we demonstrate simultaneous quasi-static and impedimetric based signal detection suggesting that FET’s could be used to readout passive membrane properties in addition to secreted charge.

Focal release of cytotoxic proteins by eosinophils onto the target surface plays an important role in parasite killing. Degranulation was stimulated by intracellular application of calcium and guanosine 5'-3-O-(thio)triphosphate via the recording patch pipette or via streptolysin-O permeabilization. Exocytotic fusion was monitored by capacitance measurements, whereas release of fluorescent weak bases, which accumulate selectively within eosinophil granules, was followed by fluorescence imaging. Several distinct types of granule fusion events were directly observed by simultaneous capacitance and fluorescence measurements. These are fusion of a single granule with the plasma membrane, intracellular granule-granule fusion, fusion of large compounds of pre-fused granules with the plasma membrane (compound exocytosis), and sequential fusion of granules to granules previously fused to the plasma membrane. Extensive granule-granule fusion was also observed by electron microscopy of permeabilized cells. All these fusion mechanisms contribute to focal release. The coexistence of distinct modes of exocytosis suggests that their regulation may modulate effector functions of eosinophils during helminth infection and allergic response.

We have studied exocytosis in rat peritoneal mast cells by cell-attached patch amperometry. Step increases in capacitance were accompanied by typical amperometric spikes due to the release of 5-hydroxytryptamine (serotonin), indicating exocytosis of typical mast cell granules. We have measured the time course of fusion pore expansion, and correlated it with release from the granule matrix. The fusion pore of mast cell granules grows in three stages. The initial expansion of the pore occurred at a rate of 5 nS/s, and in many cases an observable amperometric foot was detected. A second, rapid expansion phase occurred with a rate as high as 1000 nS/s, coinciding with the upstroke of the amperometric spike. A third, slower phase, with a rate of 5 nS/s, completed the final expansion of the fusion pore. These data reveal the very late stages in the exocytotic process, and demonstrate that the size of the fusion pore does not limit release during the upstroke of the amperometric spike or during the final, slow expansion that occurs during for the decay of the amperometric spike.

In chromaffin cells, exocytosis of single granules and properties of the fusion pore — the first connection between vesicular lumen and extracellular space1 — can be studied by cell-attached patch amperometry2, 3, which couples patch-clamp capacitance measurements4, 5, 6, 7 with simultaneous amperometric recordings of transmitter release8, 9. Here we have studied exocytosis of single chromaffin granules and endocytosis of single vesicles in cell-free inside-out membrane patches by patch capacitance measurements and patch amperometry. We excised patches from chromaffin cells by using methods developed for studying properties of single ion channels10. With low calcium concentrations in the pipette and bath, the patches showed no spontaneous exocytosis, but exocytosis could be induced in some patches by applying calcium to the cytoplasmic side of the patch. Exocytosis was also stimulated by calcium entry through the patch membrane. Initial conductances of the fusion pore were undistinguishable in cell-attached and excised patch recordings, but the subsequent pore expansion was slower in excised patches. The properties of exocytotic fusion pores in chromaffin cells are very similar to those observed in mast cells and granulocytes. Excised patches provide a tool with which to study the mechanisms of fusion pore formation and endocytosis in vitro.

Nanobiotechnology is a field that utilizes the techniques of nano- and microfabrication to study biosystems or to use biological material and principles to build new devices. As an example we discuss the development of a nanofabricated electrochemical detector array that reveals the spatio-temporal dynamics of exocytosis in single chromaffin cells. In a quantal release event a single vesicle fuses with the plasma membrane releasing its contents through the fusion pore. The time-resolved amperometric currents measured by the individual electrodes detecting different fractions of the released molecules allow determination of the time course as well as localization of quantal events. Such a device may be applicable to study the correlation of exocytotic events with signalling events that could be simultaneously monitored by fluorescence microscopy.

The release of neurotransmitters into the extracellular space is an important drug target and crucial for molecular manipulation. Amperometric measurements provide details about the amount and kinetics of transmitter release in single quantal events. However, amperometric spikes vary from cell to cell even under the same condition; therefore, a large number of release events must be analyzed to achieve the statistical significance. We present CMOS chips with 100 or 1,024 active electrodes with surface modifications for amperometry measurements. To enable amperometry measurements, polarizable electrodes such as platinum are patterned on the original aluminum metal contact on the chip. To prevent potential current leakage, a new shifted electrode design was used to enhance the reliability of post-fabrication. Platinum electrode material is deposited on the aluminum contact extending onto the periphery area and further insulated to redefine the openings of the active electrodes by lithography and sputtering. The insulation of the surface is performed by lithography using photoresist. The 100 sensor array was validated by simultaneous recording from all electrodes at 1MS/s using dopamine injection and recordings from live chromaffin cells cultured on-chip. A new chip with 1024 working electrodes capable of bi-directional current recordings was developed for fast scan cyclic voltammetry. Positive as well as negative currents are measured in the same working manner, and a current mirror and a biased cascode transistor ensure a gain error

The SNARE (Soluble NSF Attachment protein REceptor) complex, which in mammalian neurosecretory cells is composed of the proteins synaptobrevin 2 (also called VAMP2), syntaxin, and SNAP-25, plays a key role in vesicle fusion. In this review, we discuss the hypothesis that, in neurosecretory cells, fusion pore formation is directly accomplished by a conformational change in the SNARE complex via movement of the transmembrane domains.

Cell membranes behave like electrical capacitors and changes in cell capacitance therefore reflect changes in the cell area. Monitoring capacitance can thus be used to study dynamic cellular phenomenon involving rapid changes in cell surface, such as exo- and/or endocytosis. In this review focus is on the use of capacitance techniques to study exocytosis in human neutrophils. We compare the whole-cell and the cell-attached capacitance techniques, and we review the complete literature dealing with capacitance measurements in human neutrophils.

Elevated intracellular Ca2+ concentrations [Ca2+] appear to be a rather universal trigger of massive membrane capacitance increases, presumably reflecting exocytosis of tiny vesicles (Borgonovo et al., 2002). The [Ca2+] required to stimulate this response is very high. In this issue Yaradanakul et al. (see p. 29) report experiments with baby hamster kidney (BHK) cells expressing the Na+/Ca2+ exchanger NCX1. In whole-cell patch clamp experiments, using an intracellular solution with high (40 mM) Na+ concentration and a Na+-free extracellular solution, switching extracellular [Ca2+] from very low (in the presence of 0.5 mM EGTA) to 2 mM is a new trick that makes it work. The massive Ca2+influx that is produced (mediated bv the Na+/Ca2+ exchanger running in reverse) raises intracellular free [Ca2+] to ∼200 μM, which triggers a pronounced capacitance increase. The response is only partially inhibited when ATP is replaced by the nonhydrolyzable analogue AMPPNP. A Ca2+-dependent capacitance increase, distinct from secretory granule exocytosis, was first discovered in rat peritoneal mast cells (Almers and Neher, 1987) and was subsequently reported to occur in many other cell types (Lindau et al., 1993; Coorssen et al., 1996; Oberhauser et al., 1996; Xu et al., 1998; Borgonovo et al., 2002). The very high [Ca2+] increase required to induce the response in BHK cells agrees with the previously reported requirement for intracellular [Ca2+] exceeding 100 μM to induce corresponding capacitance increases in many other cells—though the response in mast cells was apparent already at ∼3 μM free intracellular [Ca2+] (Almers and Neher, 1987). Phosphoinositides have for many years been implicated to play a significant role in regulated exocytosis (Eberhard et al., 1990; Hay et al., 1995; Martin, 2001; Wenk and De Camilli, 2004). Yaradankul et al. (2008) present detailed studies on the role of phosphoinositides in the response that is triggered by high [Ca2+] in BHK cells. The Ca2+ influx activates PI(4,5)P2 breakdown but phosphoinositide metabolism turns out to be neither sufficient nor necessary for the membrane-fusion response. Activation of PI(4,5)P2 breakdown in the absence of a sufficiently high [Ca2+] increase does not stimulate fusion and PLC inhibitors as well as peptides binding PI(4,5)P2 do not interfere with the activation of the Ca2+ influx–induced fusion response. These results indicate that the regulation of this fusion response is quite different from what has been reported for hormone release from neuroendocrine cells, where PI(4,5)P2 appears to have a role in the priming as well as the fusion (Eberhard et al., 1990; Hay et al., 1995; Martin, 2001). What membrane compartment(s) could cause the observed massive capacitance changes? The usual assumption is that these changes reflect an increase in membrane area due to fusion of a large number of small vesicles that are not resolved as individual capacitance steps. Alternatively, for a membrane capacitance increase the plasma membrane thickness would have to decrease or its effective dielectric constant would have to increase dramatically. At present there is no evidence that such changes could occur on the required scale. The size of the response, however, could also not be explained by the vesicle numbers seen in electron micrographs (Yaradanakul et al., 2008). However, in thin sections such small vesicles might be lost, and a mechanism involving vesicle fusion still appears the most likely. Indeed, previous work identified the protein desmoyokin-AHNAK as a marker of the vesicles underlying this exocytotic response and which were named enlargosomes (Borgonovo et al., 2002). In chromaffin cells the fusion of microvesicles that is not associated with catecholamine release is, in contrast to chromaffin granule exocytosis, not sensitive to tetanus toxin (TeTx) as well as Botulinum neurotoxins E, D, A, and C1 (Xu et al., 1998). Fusion of enlargosomes in PC12 cells is also TeTx insensitive (Kasai et al., 1999; Borgonovo et al., 2002). It would be interesting to explore if the response is also toxin insensitive in BHK cells. If that were the case, exocytosis of enlargosomes could be independent of phosphoinositide turnover. In the second paper on the pair of articles, Wang and Hilgemann (see p. 51) extend these studies to the rat basophilic leukemia (RBL) mast cell line. Following serotonin loading, the size of secretory granules increases dramatically in RBL cells (Williams et al., 1999). Exocytosis was stimulated by addition of the Ca2+ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Extending the recently developed method of patch amperometry (Albillos et al., 1997; Dernick et al., 2005) to giant patches, the authors demonstrate that large capacitance steps, reflecting fusion of these granules with the plasma membrane, are associated with amperometrically detected serotonin release. In addition to these discrete capacitance steps, capacitance changes that cannot be resolved as discrete steps are observed—and which are not associated with serotonin release, consistent with previous observations in peritoneal mast cells (Oberhauser et al., 1996). The authors then proceed to explore the underlying mechanism(s) in excised giant patch capacitance measurements, which were performed on cells pretreated with latrunculin A to disrupt the cytoskeleton. Occasionally, but not reproducibly, exocytosis of large secretory granules associated with serotonin release was recorded by patch amperometry also in this configuration. The basis for the variability most likely is that when the patch is excised, the giant granules that are formed by serotonin loading do not stay docked to the cytoplasmic face of the giant patch but are left behind in the cell, tethered to the cytoskeleton. In giant excised-patch recordings, application of high [Ca2+] from a puffer pipette stimulates massive exocytosis. These experiments were performed on patches from cells that were not preloaded with serotonin. In these cells secretory granules containing mediators such as histamine or hexosaminidase exist but they are small. It is therefore uncertain how much of the response is due to secretory granules and how much is due to microvesicles distinct from the secretory granules. Surprisingly, the authors find almost complete inhibition of the capacitance response following TeTx treatment. Because rat synaptobrevin-1 is not toxin sensitive (Schiavo et al., 2000), the result suggests that the response is mediated by fusion of vesicles containing synaptobrevin-2 or cellubrevin. This is important because, though exocytosis of mast cell secretory granules is thought not to involve TeTx-sensitive synaptobrevin-2 or cellubrevin, recent experiments suggest that different subpopulations of vesicles undergoing regulated exocytosis in mast cells may involve different VAMP family members (Puri and Roche, 2008). The results of Wang and Hilgemann (2008) suggest that, in contrast to other cell types, exocytosis of RBL vesicles that fuse in response to high intracellular [Ca2+] is mediated by a TeTx-sensitive VAMP family member. As for the BHK cells, vesicle fusion in RBL cells appears to be independent of phosphoinositide metabolism. However, experiments with wortmannin/adenosine, which inhibits PI(3) and PI(4) kinases, point to a possible role in priming. Using a technique that inflates cells, such that the plasma membrane detaches from the vesicles bound to the cytoskeleton (Solsona et al., 1998), Wang and Hilgemann further show that the recovery of the capacitance response after reshrinking the cells is markedly reduced following wortmannin/adenosine treatment, suggesting that phosphoinositdes may play a role in priming of these vesicles after making contact with the plasma membrane, consistent with the expected role of PI(4,5)P2 in the priming of secretory vesicles in neuroendocrine cells (Hay et al., 1995; Gong et al., 2005; Milosevic et al., 2005). It still remains to be determined what vesicle type gives rise to the large capacitance changes stimulated by [Ca2+] in the hundreds of micromolar range, and if these are at all similar for all the different cell types. The differential TeTx sensitivity in different cell types suggests a quite complex picture.

Substance P and other polycationic peptides are thought to stimulate mast cell degranulation via direct activation of G proteins. We investigated the ability of extracellularly applied substance P to translocate into mast cells and the ability of intracellularly applied substance P to stimulate degranulation. In addition, we studied by reverse transcription–-PCR whether substance P-specific receptors are present in the mast cell membrane. To study translocation, a biologically active and enzymatically stable fluorescent analogue of substance P was synthesized. A rapid, substance P receptor- and energy-independent uptake of this peptide into pertussis toxin-treated and -untreated mast cells was demonstrated using confocal laser scanning microscopy. The peptide was shown to localize preferentially on or inside the mast cell granules using electron microscopic autoradiography with 125I-labeled all-D substance P and 3H-labeled substance P. Cell membrane capacitance measurements using the patch-clamp technique demonstrated that intracellularly applied substance P induced calcium transients and activated mast cell exocytosis with a time delay that depended on peptide concentration (delay of 100–500 s at concentrations of substance P from 50 to 5 μM). Degranulation in response to intracellularly applied substance P was inhibited by GDPβS and pertussis toxin, suggesting that substance P acts via G protein activation. These results support the recently proposed model of a receptor-independent mechanism of peptide-induced mast cell degranulation, which assumes a direct interaction of peptides with G protein α subunits subsequent to their translocation across the plasma membrane.

We have studied exocytosis of single small granules from human neutrophils by capacitance recordings in the cell-attached configuration. We found that 2.2% of the exocytotic events were flickers. The flickers always ended with a downward step. This indicates closing of the fusion pore. During flickering, the fusion pore conductance remained below 1 nS, and no net membrane transfer was detectable. After fusion pore expansion beyond 1 nS the pore expanded irreversibly, leading to rapid full incorporation of the granule/vesicle into the plasma membrane. Following exocytosis of single granules, a capacitance decrease directly related to the preceding increase was observed in 7% of the exocytotic events. This decrease followed immediately after irreversible pore expansion, and is presumably triggered by full incorporation of the vesicle into the patch membrane. The capacitance decrease could be interpreted as endocytosis triggered by exocytosis. However, the gradual decrease could also reflect a decrease in the “free” patch area following incorporation of an exocytosed vesicle. We conclude that non-stepwise capacitance changes must be interpreted with caution, since a number of factors go into determining cell or patch admittance.

Using planar junctions between the conducting polymer PEDOT:PSS and various electrolytes, it is possible to inject common ions and directly observe their transit through the film. The 1D geometry of the experiment allows a straightforward estimate of the ion drift mobilities.

article i nfo Background: Neurotransmitters, neuropeptides and hormones are released from secretory vesicles of nerve terminals and neuroendocrine cells by calcium-activated exocytosis. A key step in this process is the forma- tion of a fusion pore between the vesicle membrane and the plasma membrane. Exocytotic fusion leads to an increase in plasma membrane area that can be measured as a proportional increase in plasma membrane capacitance. Scope of review: High resolution capacitance measurements in single cells, nerve terminals and small mem- brane patches have become possible with the development of the patch clamp technique. This review dis- cusses the methods of whole cell patch clamp capacitance measurements and their use in conjunction with voltage clamp pulse stimulation and with stimulation by photorelease of caged calcium. It also discusses patch capacitance measurements for the study of single exocytotic events and fusion pore properties in neu- roendocrine cells and nerve terminals. Major conclusions: Capacitance measurements provide high resolution information on the extent and time course of fusion for the characterization of vesicle pools and the kinetics of exocytosis. They allow the char- acterization of the mode of fusion including distinction of single vesicle full fusion, transient kiss-and-run fu- sion or multivesicular compound exocytosis. Furthermore, measurement of fusion pore conductances and their dynamic behavior has enabled the characterization of fusion pore properties in a way that resembles the characterization of ion channel function through single channel recordings. General significance: The combination of patch clamp capacitance measurements with pharmacological and molecular manipulations of exocytosis is emerging as a powerful approach to investigate the molecular mechanisms of calcium-activated exocytotic fusion pore formation. This article is part of a Special Issue enti- tled Biochemical, biophysical and genetic approaches to intracellular calcium signalling.

Neurotransmitter release is modulated by many drugs and molecular manipulations. We present an active CMOS-based electrochemical biosensor array with high throughput capability (100 electrodes) for on-chip amperometric measurement of neurotransmitter release. The high-throughput of the biosensor array will accelerate the data collection needed to determine statistical significance of changes produced under varying conditions, from several weeks to a few hours. The biosensor is designed and fabricated using a combination of CMOS integrated circuit (IC) technology and a photolithography process to incorporate platinum working electrodes on-chip. We demonstrate the operation of an electrode array with integrated high-gain potentiostats and output time-division multiplexing with minimum dead time for readout. The on-chip working electrodes are patterned by conformal deposition of Pt and lift-off photolithography. The conformal deposition method protects the underlying electronic circuits from contact with the electrolyte that covers the electrode array during measurement. The biosensor was validated by simultaneous measurement of amperometric currents from 100 electrodes in response to dopamine injection, which revealed the time course of dopamine diffusion along the surface of the biosensor array. The biosensor simultaneously recorded neurotransmitter release successfully from multiple individual living chromaffin cells. The biosensor was capable of resolving small and fast amperometric spikes reporting release from individual vesicle secretions. We anticipate that this device will accelerate the characterization of the modulation of neurotransmitter secretion from neuronal and endocrine cells by pharmacological and molecular manipulations of the cells.

Fusion of neurosecretory vesicles with the plasma membrane is mediated by SNARE proteins, which transfer a force to the membranes. However, the mechanism by which this force transfer induces fusion pore formation is still unknown. The neuronal vesicular SNARE protein synaptobrevin 2 (syb2) is anchored in the vesicle membrane by a single C terminal transmembrane (TM) helix. In coarse grain molecular dynamics simulations self-assembly of the membrane occurred with the syb2 TM helix inserted as expected from experimental data. The free energy profile for the position of the TM domain in the membrane was determined applying harmonic potentials to the peptide in its unbiased position, pulling it towards new biased equilibrium positions. The energy profile determined in this way predicts the energy landscapes for pulling syb2 towards the extravesicular side as expected for SNARE complex zippering. Applying a constant pulling force of 160 pN detaches the synaptobrevin C terminus from the vesicle's inner leaflet lipid head groups within ∼100ns and pulls the C terminus deeper into the membrane. This C terminal movement should occur on the physiological millisecond time scale at ∼120 pN force. It is facilitated and hindered by specific mutations in parallel with experimentally observed facilitation and inhibition of fusion. These results suggest a mechanism where fusion pore formation is induced by movement of the charged syb2 C terminus into the hydrophobic core of the membrane in response to the force generated by C terminal zippering of the SNARE complex. This displacement of the charged C terminus is expected to destabilize the membrane providing a plausible pathway to fusion pore formation. Supported by NIH grants R01GM085808, R21NS072577, EDICT Project grant 201924, an MRC fellowship and the Wellcome Trust.

We have developed and tested transparent microelectrode arrays capable of simultaneous amperometric measurement of oxidizable molecules and fluorescence imaging through the electrodes. Surface patterned microelectrodes were fabricated from three different conducting materials: Indium-tin-oxide (ITO), nitrogen-doped diamond-like carbon (DLC) deposited on top of ITO, or very thin (12 - 17 nm) gold films on glass substrates. Chromaffin cells loaded with lysotracker green or acridine orange dye were placed atop the electrodes and vesicle fluorescence imaged with total internal reflection fluorescence (TIRF) microscopy while catecholamine release from single vesicles was measured as amperometric spikes with the surface patterned electrodes. Electrodes fabricated from all three materials were capable of detecting amperometric signals with high resolution. Unexpectedly, amperometric spikes recorded with ITO electrodes had only about half the amplitude and about half as much charge as those detected with DLC or gold electrodes, indicating that the ITO electrodes are not as sensitive as gold or DLC electrodes for measurement of quantal catecholamine release. The lower sensitivity of ITO electrodes was confirmed by chronoamperometry measurements comparing the currents in the presence of different analytes with the different electrode materials.